Methods for differentiation

ABSTRACT

Described herein are methods relating to the differentiation of stem cells to more differentiated phenotypes, e.g. to terminally differentiated cell types and/or precursors thereof. In some embodiments, the methods relate to contacting the stem cells with differentiation factors and halting the cell cycle, thereby increasing the rate of differentiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 62/010,244 filed Jun. 10, 2014, the contentsof which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with federal funding under Grant No. GM26875awarded by the National Institutes of Health. The U.S. government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 28, 2015, isnamed 002806-081571-PCT_SL.txt and is 314,859 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods of differentiation,e.g. differentiating stem cells.

BACKGROUND

As cells differentiate during embryonic development, they progressthrough a sequence of events, starting with stem cells, to intermediatecell types, and finally, to terminally differentiated cell types. Thisdifferentiation process can be induced in vitro. However, in vitrodifferentiation can be a time-consuming process with a significant lagtime before differentiated cells are obtained.

SUMMARY

Described herein are methods relating to the inventors' discovery thatearly and simultaneous inhibition of the cell cycle as well as theintroduction of differentiation factors can significantly reduce thetime required for cells to differentiate in vitro.

In one aspect, described herein is a method of differentiating a stemcell, the method comprising: i) contacting the stem cell with one ormore ectopic differentiation factors; and ii) inhibiting the cell cycleof the stem cell; wherein steps i) and ii) occur within 15 days of eachother.

In some embodiments, steps i) and ii) occur within 14 days of eachother. In some embodiments, steps i) and ii) occur within 13 days ofeach other. In some embodiments, steps i) and ii) occur within 12 daysof each other. In some embodiments, steps i) and ii) occur within 11days of each other. In some embodiments, steps i) and ii) occur within10 days of each other. In some embodiments, steps i) and ii) occurwithin 9 days of each other. In some embodiments, steps i) and ii) occurwithin 8 days of each other. In some embodiments, steps i) and ii) occurwithin 7 days of each other. In some embodiments, steps i) and ii) occurwithin 6 days of each other. In some embodiments, steps i) and ii) occurwithin 5 days of each other. In some embodiments, steps i) and ii) occurwithin 4 days of each other. In some embodiments, steps i) and ii) occurwithin 3 days of each other. In some embodiments, steps i) and ii) occurwithin 2 days of each other. In some embodiments, steps i) and ii) occurwithin 24 hours of each other. In some embodiments, steps i) and ii)occur simultaneously.

In some embodiments, the differentiation factor is a terminaltranscription factor. In some embodiments, the terminal transcriptionfactor is selected from Table 1.

In some embodiments, the cell cycle is inhibited by one or more of thefollowing: reducing or removing growth factors; reducing serum levels;reducing serum levels below 5%; contacting the cell with a PI3Kinhibitor; contacting the cell with an E2F family transcription factorinhibitor; contacting the cell with a Myc inhibitor; contacting the cellwith a MAPK inhibitor; contacting the cell with a MEK1/2 inhibitor;contacting the cell with a CDK inhibitor; contacting the cell with an Idinhibitor; contacting the cell with a Rb agonist; contacting the cellwith a Ink family agonist; contacting the cell with a Cip/Kip familyagonist; and culturing the cell in a media lacking a factor selectedfrom the group consisting of: LIF; Bmp; Fgf; Activin; or TGFβ. In someembodiments, the PI3K inhibitor is LY294002. In some embodiments, theE2F transcription factor inhibitor is HLM006474. In some embodiments,the Myc inhibitor is JQ1 or 10058-F4. In some embodiments, the MAPKinhibitor is PD98059. In some embodiments, the CDK inhibitor is a CDK4or CDK2 inhibitor. In some embodiments, the CDK inhibitor is p16, p15,p18, or p19. In some embodiments, the CDK inhibitor is p21, p27, or p57.

In some embodiments, the stem cell is an embryonic stem cell.

In some embodiments, steps i) and ii) result in a population of cellscomprising one or more terminally-differentiated cell types. In someembodiments, steps i) and ii) result in a population of cells comprisingno more than 2 terminally-differentiated cell types. In someembodiments, steps i) and ii) result in a population of cells of whichat least 50% are terminally-differentiated cells. In some embodiments,steps i) and ii) result in a population of cells of which at least 60%are terminally-differentiated cells. In some embodiments, steps i) andii) result in a population of cells of which at least 70% areterminally-differentiated cells. In some embodiments, steps i) and ii)result in a population of cells of which at least 80% areterminally-differentiated cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict an illustrative summary of theproliferation/differentiation network adapted for ES cells. FIG. 1Adepicts a schematic reconstructed summary of main network components andinteractions in normal somatic cycling cells. Extracellular growthfactors (GFs) either purified or in serum activate downstream signalingpathways (PI3K, MAPK), which then trigger a transcriptional activation(Myc, E2F) that drives the core cell cycle machinery (Cyclins andCyclin-dependent kinases). For a full explanation and justification ofthe summary see FIGS. 7A-7C. The cellular behavior associated with thismodel is normal oscillatory cycling. FIG. 1B depicts a schematic ofchanges to the network that occur during terminal division arrest anddifferentiation. Cells switch to insulin signaling for survival andgrowth, and shut down cycling activity. Terminal transcription factorsbecome fully active, leading to complete differentiation. FIG. 1Cdepicts a schematic composite adaptation of network for ES cells. EScells are normally maintained by LIF and high serum or LIF and Bmp4.This hyper-activates PI3K, Myc, E2F, CDK2, and Id family activities.Meanwhile, MAPK, CDK4, p16 family, p21 family, and Rb family activitiesare highly suppressed. This leads to an ultra-rapid proliferation andshort G1 phase.

FIGS. 2A-2B demonstrate that gfactor/serum reduction drives a directterminal skeletal muscle differentiation program. FIG. 2A depicts adifferentiation time course of ES cells to skeletal muscle when exposedto low serum vs high serum conditions. MyoD overexpression was initiallyinduced with tamoxifen starting at Day −1 for 24 hours under ESconditions. LIF was removed at Day 0. Low serum was then initiated atdifferent starting times beginning with Day 0. Cells were then fixed andimmunostained for myosin heavy chain (MHC) expression. Differentiatingcells in high serum were continuously split to prevent overgrowth. FIG.2B depicts a comparison of differentiation efficiency generated by twotypes of defined media (N2B27) and 20% KOSR, low serum media (2% horseserum+insulin), and high serum media (15% FBS). Cells were removed fromstandard ES media (LIF and serum) and incubated in the specified mediastarting from Day 0 to Day 4. Continuation of LIF (1000 U/ml) oraddition of Bmp4 (10 ng/ml) results in a strong block to muscledifferentiation.

FIG. 3 demonstrates that growth factor/serum withdrawal drives acondensed gene expression program of early Pax3 expression andsubsequent MyoG expression, but does not affect kinetics of Oct4 orNanog loss. Gene expression time courses of differentiation over 7 days.In this experiment, low serum was initiated at Day 0, one day afterinduction of MyoD expression in ES media, and maintained over the courseof differentiation. The rate of decline of Oct4 and Nanog mRNA levelsare not strongly affected by cell cycle inhibition. In the muscleregulatory hierarchy, only Pax3 and MyoG are strongly upregulated.Upregulation of additional terminal markers of skeletal muscle terminaldifferentiation can be found in FIG. 9. Ct values were normalized toglyceraldehyde 3-phosphate dehydrogenase (GADPH). Error values reflectS.E.M. (n=3).

FIG. 4 demonstrates that specific cell cycle inhibitors have stage andcondition-specific effects on Pax3 and MyoG expression. Time courses ofdifferentiation were run for 7 days similarly to the previous growthfactor withdrawal experiments. MyoD was induced at Day −1, and LIF wasremoved at Day 0. Cells were kept in either high serum (15% FBS) or lowserum (2% HS+Insulin) media throughout the time course. Drug treatmentswere applied continuously at the designated concentrations for theentire duration of the time course, with daily media change. mRNAexpression levels were measured by qRT-PCR. Pax3 was measured on Day 3(since it is upregulated early) and MyoG was measured on Day 7 (since itis upregulated late). All measurements are relative to a DMSO controlfor the specific condition (i.e. low serum values are still higher thanin high serum). L=lethal; drug had strong anti-survival effects so nodata was collected. Error bars indicate S.E.M. (n=2).

FIG. 5 demonstrates that in an unguided or heterogeneous differentiationsetting, growth factor/serum withdrawal upregulates the expression ofnumerous genes associated with differentiated cell types. Geneexpression time courses of unguided (without MyoD) differentiation afterrelease from ES cell media at Day 0. Low or high serum was applied atDay 0 for the full time course. Upregulated factors include Dll1, Sox6,GATA1, PPARγ, Sox9, Runx2, Mitf, Sox17, and Nkx2.2 (continued in FIG.10). These genes are involved in the differentiation of multiple celltypes, including neurons, chondrocytes, erythrocytes, adipocytes,cardiomyocytes, osteoblasts, melanocytes, and beta-cells. Error valuesindicate S.E.M. (n=2).

FIG. 6 demonstrates that use of network-based cell cycle manipulationmay provide an alternative strategy to generating terminal cell typesthat is more efficient than recapitulating embryogenesis. (Top Box)Analytical scheme of how cell cycle states drive activation of aterminal transcription factor. Without wishing to be bound by theory, itis contemplated that in the MyoD overexpression system, transitions fromthe 3 states of ES to somatic cycling to a terminally differentiatedstate influence the subsequent activation of MyoD and hence theprogression of differentiation. The first transition is correlated withPax3 activation and the second transition is correlated with MyoGactivation. Accelerating the cell cycle transitions accelerates thedifferentiation process. (Lower Box) Current strategies ofdifferentiating ES cells into cells involves growth-factor basedrecapitulation of embryogenesis. The alternative strategy describedherein (and shown in the case of skeletal muscle), is to artificiallyinduce differentiation by accelerating cell cycle inhibition combinedwith addition of a terminal transcription factor. Since ES cells aresusceptible to cell cycle-related pathways, this permits a faster, moreefficient differentiation.

FIGS. 7A-7C depict schematics of cell cycle control. Schematics areadapted from Morgan, D.O. The cell cycle: principles of control. Sinauerassociates, Sunderland, 2007 p. 207-210; which is incorporated byreference herein in its entirety. 1. Growth factors (GFs)/mitogens arereceived in G1 before the restriction point 2. The immediate-earlyresponse genes are initiated in response to mitogens. Myc then inducesCyclin D transcription and E2F activity. Entry into S-phase by Mycrequires E2F activity. Myc also can induce Inhibitor of differentiation(Id) family proteins. 3. E2F activity reciprocally promotes Mycactivity. Box represents that Myc and E2F share a transcriptionalfunction to activate pro-proliferative genes. 4. MyoD interacts withG1/S transition components in several ways. MyoD directly interacts withCyclin D/Cdk4, suppressing its activity. MyoD is also phosphorylated byCDKs leading to its ubiquitination and destruction. Rb interacts withMyoD as a co-factor on DNA. Finally, MyoD transcriptionally increasesthe expression of Rb RNA. 5. Grouped together are family members of theInk4 CDK inhibitors, Cip/Kip CDK inhibitors, Rb pocket proteins (Rb1,p107, p130), Inhibitor of DNA binding (Id) proteins, and E2Ftranscription factors. It is recognized that individual family membershave unique features, however this complexity is not represented in thismodel. 6. In mES cells, LIF drives Myc activity through Jak/Statsignaling. LIF also increases PI3K activity. 7. Bmp signaling leads toupregulation of Inhibitor of differentiation (Id) proteins, which arepredicted to inhibit Rb and MyoD 8. c-Myc has been observed to bind Rbin vitro, but not in vivo. On the other hand, p107 does interact withMyc in vivo. In mES cells, Myc is expressed at high levels and may beinhibiting Rb family members. 9. In certain cases, Myc or E2Foverexpression leads to a reduction in Cyclin D1 levels. It iscontemplated that the extra activation of these factors in mES cellsdecreases Cyclin D1 levels. 10. c-Myc has been suspected toindependently suppress myod outside of cell transformation.

FIG. 8 depicts microscopy images demonstrating the morphology ofdifferentiated muscle myotubes stained for myosin heavy chain andsarcomeric a-actinin. FIG. 8A depicts myosin heavy chain staining is inwhite coloring in the cytoplasm. Myotubes exhibit an elongated,multinucleated morphology. Arrows point to nuclei in a binucleatemyotube. This particular image was taken on day 8 of the low serum timecourse started on day 0. FIGS. 8B-8D depict sarcomeric a-actininstaining of myofibrillar striations in differentiated myotubes. Theseparticular images were taken on day 13.

FIG. 9 demonstrates additional muscle genes upregulated by growthfactor/serum removal. Microarray data confirm expression of skeletalmuscle genes during MyoD differentiation by low serum Desmin (Des),Myosin light chain (Myl1), skeletal muscle actin (Acta1), troponin(Tnnc1), tropomyosin (Tpm1), Mirk/Dyrk1b, and titin (Ttn) getupregulated during the differentiation time course. In this experiment,MyoD overexpression is activated at Day −1 in ES media. LIF is thenremoved at Day 0 and the media is switched to low serum.

FIG. 10 demonstrates additional time courses of lineage-specific factorsdifferentiated by low serum and LIF removal (no MyoD).

FIGS. 11A-11B depict spinal motor neurons. FIG. 11A demonstrates rapidneuron formation from iNIL ES cells Beta-3 tubulin (terminal neuronmarker) mRNA expression after 4 days in media of different growth factorcontent. FIG. 11B depicts a time course of gene expression for neuronallineage factors and motor neuron terminal differentiation markers ingrowth factor-free (N2B27) media.

FIGS. 12A-12B depict cardiomyocytes. FIG. 12A demonstrates rapidkinetics of cardiomyocyte formation from GATA5-overexpressing ES cells.Cardiac troponin (terminal cardiomyocyte marker) mRNA expression after 4days in media of different growth factor content. FIG. 12B depicts atime course of gene expression for cardiac lineage factors andcardiomyocyte terminal differentiation markers in growth factor-free(N2B27) media.

FIGS. 13A-13B depict hepatoblast-like cells. FIG. 13A demonstrateshepatoblast formation from Hnf4α-overexpressing ES cells.Alpha-fetoprotein mRNA expression after 4 days in media of differentgrowth factor content. FIG. 13B depicts a time course of gene expressionfor hepatic lineage factors and hepatocyte markers in growth factor-free(N2B27) media.

FIG. 14 depicts pictures of spinal motor neurons. Top: Phase contrastimage of Day 5 neurons differentiated in growth factor-free N2B27 media.Bottom: Same neurons immunostained with antibody against neuronalbeta3-tubulin (Tubb3).

FIG. 15 depicts an image of a cluster of cardiomyocytes differentiatedafter 9 days beating in the dish.

FIG. 16 depicts pictures of hepatoblast-like cells. Top: Phase contrastimage of Day 5 hepatoblast-like cells differentiated in growthfactor-free N2B27 media. Image is crowded due to the continuedproliferation of these cells in growth factor-free media. Bottom: Samehepatoblast-like cells immunostained with antibody against alphafetoprotein (AFP

DETAILED DESCRIPTION

As described herein, the inventors's have found that inhibiting the cellcycle and introducing differentiation factors, when both steps areperformed early in the differentiation process, significantly increasesthe rate of differentiation (e.g. the differentiation of stem cells toterminally differentiated cells). In one aspect, described herein is amethod of differentiating a stem cell, the method comprising (i)contacting the stem cell with one or more ectopic differentiationfactors and (ii) inhibiting the cell cycle of the stem cell. In someembodiments, the stem cell can be an embryonic stem cell. In someembodiments, the stem cell can be an adult stem cell. In someembodiments, the stem cell can be an induced pluripotent stem cell.

Accordingly, in some embodiments, step (i) (i.e., contacting the stemcell with one or more ectopic differentiation factors) and step (ii)(i.e., inhibiting the cell cycle of the stem cell) are performed withinabout 15 days or less of each other, e.g. within about 14 days or lessof each other, within about 13 days or less of each other, within about12 days or less of each other, within about 11 days or less of eachother, within about 10 days or less of each other, within about 9 daysor less of each other, within about 8 days or less of each other, withinabout 7 days or less of each other, within about 6 days or less of eachother, within about 5 days or less of each other, within about 4 days orless of each other, within about 3 days or less of each other, withinabout 2 days or less of each other, or within about 1 day or less ofeach other. In some embodiments, steps (i) and (ii) can be performedabout simultaneously.

In some embodiments, step (i) (i.e., contacting the stem cell with oneor more ectopic differentiation factors) and step (ii) (i.e., inhibitingthe cell cycle of the stem cell) are performed within 15 days of eachother, e.g. within 14 days of each other, within 13 days of each other,within 12 days of each other, within 11 days of each other, within 10days of each other, within 9 days of each other, within 8 days of eachother, within 7 days of each other, within 6 days of each other, within5 days of each other, within 4 days of each other, within 3 days of eachother, within 2 days of each other, or within 1 day (e.g. within 24hours) of each other. In some embodiments, steps (i) and (ii) can beperformed simultaneously.

In some embodiments, steps (i) and (ii) can both be performed before thestem cell differentiates to an intermediate and/or terminallydifferentiated cell, e.g. while the stem cell still evidences a stemcell phenotype. In some embodiments, a stem cell can be anundifferentiated cell exhibiting both pluripotency (or totipotent) andcapable of self-renewal. In some embodiments, a stem cell can be a cellexpressing stem cell markers. Stem cell markers are known in the art andcan include, by way of non-limiting example, Nanog, SSEA-1, TDGF-1,Sox2, Oct4, (for further detail see, e.g., Pazhianisamy MATER METHODS2013 3:200 and Zhao et al. Molecules 2013 17:6196-6236; each of which isincorporated by reference herein in its entirety). Kits for determiningif a cell expresses stem cell markers are commercially available, e.g.Cat No. ab109884 from AbCam, Cambridge, Mass.

As used herein, “ectopic differentiation factor” refers to an ectopicagent that increases and/or promotes the process of differentiation.Ectopic differentiation factors are known in the art and can include,e.g. nucleic acids encoding polypeptides, polypeptides, small molecules,growth factors, cytokines, and the like. The identity of the ectopicdifferentiation factor will vary according to the type of differentiatedcell that is desired. Appropriate ectopic differentiation factors thatpermit the differentiation of specific differentiated cell types areknown in the art, see, e.g. Examples of various differentiation agentsare disclosed in U.S. Patent Application Serial No. 2003/0022367, orGimble et al., 1995; Lennon et al., 1995; Majumdar et al., 1998; Caplanand Goldberg, 1999; Ohgushi and Caplan, 1999; Pittenger et al., 1999;Caplan and Bruder, 2001; Fukuda, 2001; Worster et al., 2001; Zuk et al.,2001; Rosenbauer and Tenen Nature Reviews Immunology 2007 7:105-117;Hughes et al. Periodontology 2006 41:48-72; Loregger et al. Placenta2003 A:S104-110; Florini et al. Ann Rev of Physiology 1991 53:201-216;Yamamizu et al. Stem Cell Reports 2013 1:545-559; James. Scientifica2013 684736; and Mummery et al. Circulation Research 2012 111:344-358;each of which is incorporated by reference herein in its entirety.

In some embodiments, the ectopic differentiation factor can be aterminal transcription factor. As used herein, the term “terminaltranscription factor” refers to a transcription factor that promotes thedifferentiation of a stem cell and/or intermediate or partiallydifferentiated cell into a terminally differentiated cell. A cell thatis contacted with a terminal transcription factor can be contacted witheither an ectopic nucleic acid encoding a terminal transcription factorand/or an ectopic terminal transcription factor polypeptide.

Terminal transcription factors, and the differentiated phenotypes theypromote are known in the art. Non-limiting examples of terminaltranscription factors can include MyoD; MyoG, Myf5, Mrf4, Ngn family(e.g. Ngn1-3), NeuroD family (e.g. NeuroD1-3), Ascl family (e.g.Ascl1-2), Hb9, Zic1, Brn2, Mytl1, Nurr1, Lmx1a, Gata family (e.g. Gata1,2, 4, 5, 6), Tbx5, Mef2 family (e.g. Mef2a,b,c), Mesp1, Hnf/FoxA family(e.g. Hnf4α, FoxA2), Pdx1, MafA, Runx family (e.g. Runx2, Runx1t1),Mitf, Spi1, Nkx family (e.g. Nkx2.1, 2.2), C/EBP family (e.g. C/EBPα, β)Prdm family (e.g. Prdm1, 16), PPARγ, Scl, Lmo2, Ldb1, E2A, Ebf, Sox9,Hlf, Prdm5, Pbx1, Zfp37, Isl1, Lhx3, Phox2a, Fezf2, Olig family (e.g. 1and 2), Elf5, Irf2, Elf1, Tgif1, Ets1, Sox family (e.g. Sox4, 6, 9, 17),Bach2, Cdx2, Smyd1, Pax family (e.g. Pax3, 6, 7), Klf family (e.g.Klf4), basic helix-loop-helix factors. A non-limiting list of exemplaryterminal transcription factors is provided in Table 1.

TABLE 1 Exemplary Terminal Transcription Factors Exemplary AssociatedTerminal Transcription NCBI Gene ID for Differentiated Cell Type FactorHuman Gene Myoblast MyoD 4654 MyoG 4656 Myf5 4617 Mrf4 4618 Gata4 2626Tbx5 6910 Mef2a 4205 Mef2b 100271849 Mef2c 4208 Mesp1 55897 Pax7 5081Smyd1 150572 Neuron Ngn1 4762 Ngn2 63973 Ngn3 50674 NeuroD1 4760 NeuroD24761 NeuroD3 4762 Ascl1 429 Ascl2 430 Zic1 7545 Brn2 5454 Nurr1 4929Mytl1 23040 Lmx1a 4009 Hlf 3131 Zfp37 7539 Phox2a 401 Fezf2 55079 Motorneurons/Pancreatic Hb9 3110 cells Isl1 3670 Erythroid Gata1 2623 Ldb18861 Haemotopoietic and Gata2 2624 Endocrine lineages Myocardial andendodermal Gata5 140628 lineages Endodermal and Gata6 2627 Mesodermallineages Hepatocyte (also liver, Hnf4α 3172 kidney, & intestinal cells)Hepatocytes FoxA2 3170 Pancreatic Pdx1 3651 MafA 389692 Osteoblast Runx2860 Prdm5 11107 Pbx1 5087 Melanocytes Mitf 4286 Myeloid, B lymphoid Spi16688 Thyroid Nkx2.1 7080 Central Nervous System Nkx2.2 4821 AdipocytesC/EBPα 1050 Prdm16 63976 C/EBPβ 1051 PPARγ 5468 Trophoblasts, plasmacells Prdm1 639 Osteoblast, neurons, Scl 6886 haemotopoietic lineagesHaemotopoietic Lmo2 4005 Runx1t1 862 Bach2 60468 Irf2 3660 Sox4 6659Lymphocytes E2A 6929 Elf1 1997 B cells Ebf 1879 Chondrocytes Sox9 6662Motor Neurons Lhx3 8022 Oligodendrocytes Olig1 116448 Olig2 10215Epithelial lineage Elf5 2001 Klf4 9314 Myeloid Tgif1 7050 Haemotopoieticand Ets1 2113 Epithelial lineages Neurons and chondrocytes Sox6 55553Endoderm and Sox17 64321 Haemotopoietic lineages Intestinal Cdx2 1045Muscle cells Pax3 5077 Nervous system and eye Pax6 5080 cells Skeletalmuscle cells MyoD 4654 Spinal motor neurons Ngn2 63973 Isl1 3670 Lhx38022 Cardiomyocytes Gata5 140628 Hepatocytes/Hepatoblasts Hnf4α 3172

In some embodiments, the stem cell is to be differentiated to a skeletalmuscle phenotype and is contacted with the terminal transcription factorMyoD. In some embodiments, the stem cell is to be differentiated to askeletal muscle phenotype and is contacted with the terminaltranscription factor MyoD and the cell cycle is inhibited by reducing orremoving growth factors. In some embodiments, the stem cell is to bedifferentiated to a skeletal muscle phenotype and is contacted with theterminal transcription factor MyoD and the cell cycle is inhibited byculturing the cell in a media lacking a factor selected from the groupconsisting of: LIF; Bmp; Fgf; Activin; or TGFβ. In some embodiments, thestem cell is to be differentiated to a skeletal muscle phenotype and iscontacted with the terminal transcription factor MyoD and the cell cycleis inhibited by culturing the cell in a media lacking LIF.

In some embodiments, the stem cell is to be differentiated to a spinalmotor neuron phenotype and is contacted with a terminal transcriptionfactor selected from the group consisting of: Ngn2; Isl1; and Lhx3. Insome embodiments, the stem cell is to be differentiated to a spinalmotor neuron phenotype and is contacted with a terminal transcriptionfactor selected from the group consisting of: Ngn2; Isl1; and Lhx3 andthe cell cycle is inhibited by reducing or removing growth factors. Insome embodiments, the stem cell is to be differentiated to a spinalmotor neuron phenotype and is contacted with a terminal transcriptionfactor selected from the group consisting of: Ngn2; Isl1; and Lhx3 andthe cell cycle is inhibited by culturing the cell in a media lacking afactor selected from the group consisting of: LIF; Bmp; Fgf; Activin; orTGFβ. In some embodiments, the stem cell is to be differentiated to aspinal motor neuron phenotype and is contacted with a terminaltranscription factor selected from the group consisting of: Ngn2; Isl1;and Lhx3 and the cell cycle is inhibited by culturing the cell in amedia lacking LIF. In some embodiments, the stem cell is to bedifferentiated to a spinal motor neuron phenotype and is contacted withthe terminal transcription factors Ngn2; Isl1; and Lhx3. In someembodiments, the stem cell is to be differentiated to a spinal motorneuron phenotype and is contacted with the terminal transcriptionfactors Ngn2; Isl1; and Lhx3 and the cell cycle is inhibited by reducingor removing growth factors. In some embodiments, the stem cell is to bedifferentiated to a spinal motor neuron phenotype and is contacted withthe terminal transcription factors Ngn2; Isl1; and Lhx3 and the cellcycle is inhibited by culturing the cell in a media lacking a factorselected from the group consisting of: LIF; Bmp; Fgf; Activin; or TGFβ.In some embodiments, the stem cell is to be differentiated to a spinalmotor neuron phenotype and is contacted with the terminal transcriptionfactors Ngn2; Isl1; and Lhx3 and the cell cycle is inhibited byculturing the cell in a media lacking LIF.

In some embodiments, the stem cell is to be differentiated to acardiomyocyte phenotype and is contacted with the terminal transcriptionfactor Gata5. In some embodiments, the stem cell is to be differentiatedto a cardiomyocyte phenotype and is contacted with the terminaltranscription factor Gata5 and the cell cycle is inhibited by reducingor removing growth factors. In some embodiments, the stem cell is to bedifferentiated to a cardiomyocyte phenotype and is contacted with theterminal transcription factor Gata5 and the cell cycle is inhibited byculturing the cell in a media lacking a factor selected from the groupconsisting of: LIF; Bmp; Fgf; Activin; or TGFβ. In some embodiments, thestem cell is to be differentiated to a cardiomyocyte phenotype and iscontacted with the terminal transcription factor Gata5 and the cellcycle is inhibited by culturing the cell in a media lacking LIF.

In some embodiments, the stem cell is to be differentiated to ahepatocyte or hepatoblast phenotype and is contacted with the terminaltranscription factor Hnf4α. In some embodiments, the stem cell is to bedifferentiated to a hepatocyte or hepatoblast phenotype and is contactedwith the terminal transcription factor Hnf4α and the cell cycle isinhibited by reducing or removing growth factors. In some embodiments,the stem cell is to be differentiated to a hepatocyte or hepatoblastphenotype and is contacted with the terminal transcription factor Hnf4αand the cell cycle is inhibited by culturing the cell in a media lackinga factor selected from the group consisting of: LIF; Bmp; Fgf; Activin;or TGFβ. In some embodiments, the stem cell is to be differentiated to ahepatocyte or hepatoblast phenotype and is contacted with the terminaltranscription factor Hnf4α and the cell cycle is inhibited by culturingthe cell in a media lacking LIF.

In some embodiments, the ectopic differentiation factor can be apolypeptide. In some embodiments, the ectopic differentiation factor canbe a terminal transcription factor polypeptide. In some embodiments, theectopic differentiation factor can be a variant of a terminaltranscription factor polypeptide. In some embodiments, the ectopicdifferentiation factor can be a functional fragment of a terminaltranscription factor polypeptide.

As used herein, a given “polypeptide” can include the human polypeptide,as well as homologs from other species, including but not limited tobovine, dog, cat chicken, murine, rat, porcine, ovine, turkey, horse,fish, baboon and other primates. The terms also refer to fragments orvariants of a polypeptide that maintain at least 50% of the activity oreffect, e.g. transcriptional activation and/or suppression of afull-length polypeptide. Conservative substitution variants thatmaintain the activity of wildtype polypeptides will include aconservative substitution as defined herein. The identification of aminoacids most likely to be tolerant of conservative substitution whilemaintaining at least 50% of the activity of the wildtype is guided by,for example, sequence alignment with homologs or paralogs from otherspecies Amino acids that are identical between homologs are less likelyto tolerate change, while those showing conservative differences areobviously much more likely to tolerate conservative change in thecontext of an artificial variant. Similarly, positions withnon-conservative differences are less likely to be critical to functionand more likely to tolerate conservative substitution in an artificialvariant. Variants, fragments, and/or fusion proteins can be tested foractivity, for example, by transcriptional activity assays and/ordifferentiation assays.

In some embodiments, the variant is a conservative substitution variant.Variants can be obtained by mutations of native nucleotide sequences,for example. A “variant,” as referred to herein, is a polypeptidesubstantially homologous to a native or reference polypeptide, but whichhas an amino acid sequence different from that of the native orreference polypeptide because of one or a plurality of deletions,insertions or substitutions. Polypeptide-encoding DNA sequencesencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to a native or reference DNAsequence, but that encode a variant protein or fragment thereof thatretains the relevant biological activity relative to the referenceprotein, e.g., at least 50% of an activity of the wildtype polypeptide.As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters a single amino acid or asmall percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer,or 1% or fewer) of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid. Itis contemplated that some changes can potentially improve the relevantactivity, such that a variant, whether conservative or not, has morethan 100% of the activity of the wildtype polypeptide, e.g. 110%, 125%,150%, 175%, 200%, 500%, 1000% or more.

One method of identifying amino acid residues which can be substitutedis to align, for example, a human polypeptide with a homolog from one ormore non-human species. Alignment can provide guidance regarding notonly residues likely to be necessary for function but also, conversely,those residues likely to tolerate change. Where, for example, analignment shows two identical or similar amino acids at correspondingpositions, it is more likely that that site is important functionally.Where, conversely, alignment shows residues in corresponding positionsto differ significantly in size, charge, hydrophobicity, etc., it ismore likely that that site can tolerate variation in a functionalpolypeptide. The variant amino acid or DNA sequence can be at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, ormore, identical to a native or reference sequence, or a nucleic acidencoding one of those amino acid sequences. The degree of homology(percent identity) between a native and a mutant sequence can bedetermined, for example, by comparing the two sequences using freelyavailable computer programs commonly employed for this purpose on theworld wide web. The variant amino acid or DNA sequence can be at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or more,similar to the sequence from which it is derived (referred to herein asan “original” sequence). The degree of similarity (percent similarity)between an original and a mutant sequence can be determined, forexample, by using a similarity matrix. Similarity matrices are wellknown in the art and a number of tools for comparing two sequences usingsimilarity matrices are freely available online, e.g. BLASTp (availableon the world wide web at http://blast.ncbi.nlm.nih.gov), with defaultparameters set.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity of a nativeor reference polypeptide is retained. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and allelesconsistent with the disclosure. Typically conservative substitutions forone another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

In some embodiments, a polypeptide, can comprise one or more amino acidsubstitutions or modifications. In some embodiments, the substitutionsand/or modifications can prevent or reduce proteolytic degradationand/or prolong half-life of the polypeptide. In some embodiments, apolypeptide can be modified by conjugating or fusing it to otherpolypeptide or polypeptide domains such as, by way of non-limitingexample, transferrin (WO06096515A2), albumin (Yeh et al., 1992), growthhormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and/or Fcfragments (Ashkenazi and Chamow, 1997). The references in the foregoingparagraph are incorporated by reference herein in their entireties.

In some embodiments, a polypeptide as described herein can comprise atleast one peptide bond replacement. A polypeptide as described hereincan comprise one type of peptide bond replacement or multiple types ofpeptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, ormore types of peptide bond replacements. Non-limiting examples ofpeptide bond replacements include urea, thiourea, carbamate, sulfonylurea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid,para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylaceticacid, thioamide, tetrazole, boronic ester, olefinic group, andderivatives thereof.

In some embodiments, a polypeptide, as described herein can comprisenaturally occurring amino acids commonly found in polypeptides and/orproteins produced by living organisms, e.g. Ala (A), Val (V), Leu (L),Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T),Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R),and His (H). In some embodiments, a polypeptide as described herein cancomprise alternative amino acids. Non-limiting examples of alternativeamino acids include, D-amino acids; beta-amino acids; homocysteine,phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine (3-mercapto-D-valine), ornithine, citruline,alpha-methyl-alanine, para-benzoylphenylalanine, para-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine,sarcosine, and tert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylicacid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;azide-modified amino acids; alkyne-modified amino acids; cyano-modifiedamino acids; and derivatives thereof.

In some embodiments, a polypeptide can be modified, e.g. by addition ofa moiety to one or more of the amino acids that together comprise thepeptide. In some embodiments, a polypeptide as described herein cancomprise one or more moiety molecules, e.g. 1 or more moiety moleculesper polypeptide, 2 or more moiety molecules per polypeptide, 5 or moremoiety molecules per polypeptide, 10 or more moiety molecules perpolypeptide or more moiety molecules per polypeptide. In someembodiments, a polypeptide as described herein can comprise one moretypes of modifications and/or moieties, e.g. 1 type of modification, 2types of modifications, 3 types of modifications or more types ofmodifications. Non-limiting examples of modifications and/or moietiesinclude PEGylation; glycosylation; HESylation; ELPylation; lipidation;acetylation; amidation; end-capping modifications; cyano groups;phosphorylation; albumin, and cyclization. In some embodiments, anend-capping modification can comprise acetylation at the N-terminus,N-terminal acylation, and N-terminal formylation. In some embodiments,an end-capping modification can comprise amidation at the C-terminus,introduction of C-terminal alcohol, aldehyde, ester, and thioestermoieties. The half-life of a polypeptide can be increased by theaddition of moieties, e.g. PEG, albumin, or other fusion partners (e.g.Fc fragment of an immunoglobin).

In some embodiments, the polypeptide can be a functional fragment of oneof the amino acid sequences described herein. As used herein, a“functional fragment” is a fragment or segment of a polypeptide whichretains the activity, e.g. the transcriptional activity, of the wildtypepolypeptide. A functional fragment can comprise conservativesubstitutions of the sequences disclosed herein.

Alterations of the original amino acid sequence can be accomplished byany of a number of techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites permitting ligation to fragments of the nativesequence. Following ligation, the resulting reconstructed sequenceencodes an analog having the desired amino acid insertion, substitution,or deletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Khudyakov et al. “Artificial DNA: Methods andApplications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols”Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook”Springer 2000; which are herein incorporated by reference in theirentireties. In some embodiments, a polypeptide as described herein canbe chemically synthesized and mutations can be incorporated as part ofthe chemical synthesis process.

In some embodiments, a cell can be contacted with multiple ectopicdifferentiation factors, e.g. two or more terminal transcriptionfactors, or a terminal transcription factor and cytokine.

As used herein “cell cycle” refers to the series of events involving thegrowth, replication, and division of a eukaryotic cell. A “phase of acell cycle” or “cell cycle phase” refers to a distinct phase or periodof the cell cycle, such as the mitosis phase (M phase), the first gapphase (G1 phase), the DNA synthesis phase (S phase), and the second gapphase (G2 phase). A “complete cell cycle” refers to entire single cellcycle including a G1 phase, S phase, G2 phase, and an M phase. Analysisof a complete cell cycle does not require beginning at a particularphase within the cell cycle. For example, a “complete cellcycle phase”may begin with an S phase and end at completion of G1 phase, orlikewise, a “complete cell cycle phase” may begin with an M phase andend with completion of G2 phase Inhibition of the cell cycle cancomprise slowing the progression of a cell through the cycle, slowingthe progression of a cell through a particular stage of the cell cycle,and/or arresting the cell at a particular point in the cell cycle.Slowing the progression of the cell constitutes a decrease in the rateat which the cell progresses through the cell cycle.

Methods of inhibiting the cell cycle can comprise contacting the cellwith an agent that inhibits the cell cycle and/or removing an agent thatpromotes progression through the cell cycle. Agents for promoting orinhibiting cell cycle progression are known in the art. By way ofnon-limiting example, the cell cycle can be inhibited by: reducing orremoving growth factors; reducing serum levels; reducing serum levelsbelow about 5%; reducing serum levels below 5%; contacting the cell witha PI3K inhibitor; contacting the cell with an E2F family transcriptionfactor inhibitor; contacting the cell with a Myc inhibitor; contactingthe cell with a MAPK inhibitor; contacting the cell with a MEK1/2inhibitor; contacting the cell with a CDK inhibitor; contacting the cellwith an Id inhibitor; contacting the cell with a Rb agonist; contactingthe cell with a Ink family agonist; contacting the cell with a Cip/Kipfamily agonist; culturing the cell in a media lacking a factor selectedfrom the group consisting of: LIF; Bmp; Fgf; Activin; or TGFβ.

As used herein, the term “inhibitor” refers to an agent which candecrease the expression and/or activity of the targeted expressionproduct (e.g. mRNA encoding the target or a target polypeptide), e.g. byat least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80%or more, 90% or more, 95% or more, or 98% or more. The efficacy of aninhibitor, e.g. its ability to decrease the level and/or activity of thetarget, can be determined, e.g. by measuring the level of an expressionproduct of and/or the activity of the target. Methods for measuring thelevel of a given mRNA and/or polypeptide are known to one of skill inthe art, e.g. RTPCR can be used to determine the level of RNA andWestern blotting with an antibody can be used to determine the level ofa polypeptide. The activity of a target can be determined using methodsknown in the art and described herein, e.g. transcriptional activityassays. In some embodiments, the inhibitor can be an inhibitory nucleicacid; an aptamer; an antibody reagent; an antibody; or a small molecule.

As used herein, the term “agonist” refers to any agent that increasesthe level and/or activity of the target, e.g, of Rb, Ink familypolypeptides, and/or Cip/Kip family polypeptides. As used herein, theterm “agonist” refers to an agent which increases the expression and/oractivity of the target by at least 10% or more, e.g. by 10% or more, 50%or more, 100% or more, 200% or more, 500% or more, or 1000% or more.

Phosphoinositide 3-kinases are a family of related enzymes that arecapable of phosphorylating the 3 position hydroxyl group of the inositolring of phosphatidylinositol. They are also known asphosphatidylinositol-3-kinases. PI3Ks interact with the IRS (Insulinreceptor substrate) in order to regulate glucose uptake through a seriesof phosphorylation events. The phosphoinositol-3-kinase family iscomposed of Class I, II and Class III, with Class I the only ones ableto convert PI(4,5)P2 to PI(3,4,5)P3 on the inner leaflet of the plasmamembrane. As used herein, a “PI3K inhibitor” refers to an agent thatinhibits the activity of PI3K, as measured by the level ofphosphorylation of the 3 position hydroxyl group of the inositol ring ofphosphatidylinositol, or as measured by the activity and/orphosphorylation (where increased phosphorylation indicates PI3Kactivity) of molecules downstream of PI3K. Examples of such downstreammolecules are known in the art and can include, but are not limited toAKT, SGK, mTOR, GSK3β, PSD-95, S6, and 4EBP1. Methods of measuring theactivity of PI3K, directly or indirectly are well known in the art, andinclude, by way of non-limiting example determining the level ofphosphorylation of a molecule downstream of PI3K using phospho-isoformspecific antibodies, which are commercially available (e.g.anti-phospho-AKT antibody, Cat No. ab66138 Abcam, Cambridge, Mass.).Non-limiting examples of PI3K inhibitors can include LY294002; BGT226;BEZ235; PI103, PI828. wortmannin, demethoxyviridin, IC486068, IC87114,GDC-0941, perifosine, CAL101, PX-866, IPI-145, BAY 80-6946, P6503,TGR1202, SF1126, INK1117, BKM120, IL147, XL765, Palomid 529, GSK1059615,ZSTK474, PWT33597, TG100-115, CAL263, GNE-447, CUDC-907, and AEZS-136.

An inhibitor of E2F family transcription factors can be an agent thatinhibits the activity of a E2F transcription factor, as measured by thelevel of transcription of E2F targets (e.g. CCNA1, MYB, EB1, BRCA1, andTP53). Assays for E2F activity are known in the art and are commerciallyavailable (e.g. Cat. No. CCS-003L, Qiagen Valencia, Calif.).Non-limiting examples of E2F family transcription factors can includeHLM006474.

An inhibitor of Myc can be an agent that inhibits the activity and/orlevel of Myc. Myc is a transcription factor that participates in cellproliferation and DNA replication. The sequence of Myc is known in anumber of species, e.g. human Myc (NCBI Gene ID: 4609) mRNA (NCBI RefSeq: NM 002467 (SEQ ID NO: 85)) and polypeptide (NP 002458 (SEQ ID NO:86)) sequences. Myc activity can be measured, e.g. by the level oftranscription of genes activated (e.g. CDK or MNT) or suppressed (e.g.Miz1) by Myc. Assays for Myc activity are known in the art and arecommercially available (e.g. Cat. No. CCS-012L; Qiagen Valencia,Calif.). Non-limiting examples of Myc inhibitors can include JQ1;10058-F4; and CAS 403811-55-21.

An inhibitor of MAPK can be an agent that inhibits the activity of amitogen-activated protein kinase (MAPK), as measured by the level ofphosphorylation of MAPK targets (e.g. ELK1 is a substrate of ERK1 andMK2 and MK3 are targets of p38 kinases). Assays for MAPK activity areknown in the art and are commercially available (e.g. Cat. No. CS0250from Sigma-Aldrich, St. Louis, Mo.). Non-limiting examples of MAPKinhibitors can include PD98059; SB203580; SB202190; and SP600125.

An inhibitor of CDK can be an agent that inhibits the activity of acyclin-dependent kinase (CDK), as measured by the level ofphosphorylation of CDK targets (e.g. CDK2 targets Rb, p53 and E2F aresubstrates of CDK2 and RB1 and MEP50 are substrates of CDK4). Assays forCDK activity are known in the art and are commercially available (e.g.Cat. No. PV3343 Invitrogen, Carlsbad, Calif.). In some embodiments, aninhibitor of CDK can inhibit CDK4 and/or CDK2. In some embodiments, aninhibitor of CDK can specifically inhibit CDK4 and/or CDK2. Non-limitingexamples of CDK inhibitors can include p16; p15; p18; p19; p21; p27;p57; p1446A-05; PD-0332991; flavopiridol; aloisine A; AT7519; BS-181;butyrolactone I; purvalanol A; pruvalanol B; roscovitine; and WHI-P180.

An inhibitor of Id can be an agent that inhibits the level and/oractivity of inhibitor of DNA binding 1 (Id). Id forms heterodimers withhelix-loop-helix transcription factors and inhibits their activity. Thesequence of Id is known in a number of species, e.g. human Id (NCBI GeneID: 3397) mRNA (NCBI Ref Seq: NM_002165 (SEQ ID NO: 87)) and polypeptide(NP_002156 (SEQ ID NO: 88)) sequences. Id activity can be measured, e.g.by measuring the inhibition of DNA binding and/or transcriptionalactivation of helix-loop-helix transcription factors that can bind withId. Non-limiting examples of Id inhibitors can include caveolin-1.

An agonist of Rb can be an agent that increases the level and/oractivity of retinoblastoma 1 (Rb). Rb binds to and inhibits E2Ftranscription factors, thereby preventing progress through the cellcycle. The sequence of Rb is known in a number of species, e.g. human Rb(NCBI Gene ID: 5925) mRNA (NCBI Ref Seq: NM_000321 (SEQ ID NO: 89)) andpolypeptide (NP_000312 (SEQ ID NO: 90)) sequences. Rb activity can bemeasured, e.g., by measuring binding to E2F transcription factors and/ortranscription of E2F factors. Non-limiting examples of agonists of Rbcan include Rb polypeptides or agonist fragments thereof and nucleicacids encoding a Rb polypeptide.

An agonist of Ink family proteins can be an agent that increase thelevel and/or activity of Ink family proteins (e.g. INK4 family, INK4A(NCBI Gene ID: 1029 (SEQ ID NOS 91-92)), INK4B (NCBI Gene ID: 1030 (SEQID NOS 93-94)), INK4C (NCBI Gene ID: 1031 (SEQ ID NOS 95-96)), and INK4D(NCBI Gene ID: 1032 (SEQ ID NOS 97-98))). INK family proteins bind andinhibit CDK4 and CDK6. Ink protein activity can be measured bymeasuring, e.g. the activity of CDK4 and/or CDK6 as described elsewhereherein and/or binding to CDK4 and/or CDK6. Non-limiting examples ofagonists of Ink family polypeptides can include Ink family polypeptidesor agonist fragments thereof and nucleic acids encoding an Ink familypolypeptide.

An agonist of Cip/Kip family proteins can be an agent that increase thelevel and/or activity of Cip/Kip family proteins (e.g. Cip/Kip family,KIP1 (NCBI Gene ID: 1027 (SEQ ID NOS 99-100)), KIP2 (NCBI Gene ID: 1028(SEQ ID NOS 101-102)), and CIP1 (NCBI Gene ID: 1026 (SEQ ID NOS103-104))). Cip/Kip family proteins bind and inhibit CDK2. Cip/Kipprotein activity can be measured by measuring, e.g. the activity of CDK2as described elsewhere herein. Non-limiting examples of agonists ofCip/Kip family polypeptides can include Cip/Kip family polypeptides oragonist fragments thereof and nucleic acids encoding an Cip/Kip familypolypeptide.

In some embodiments, inhibition of the cell cycle can be accomplished byremoving and/or reducing the level of growth and/or signaling factors,e.g. by culturing the cell in media lacking or having reduced levels ofone or more growth and/or signaling factors that promote the cell cycle.Non-limiting examples of such growth and/or signaling factors caninclude LIF (e.g., NCBI Gene ID: 3976 (SEQ ID NOS 105-106)), Bmp (e.g.NCBI Gene ID: 649 (SEQ ID NOS 107-108), 650 (SEQ ID NOS 109-110), 651(SEQ ID NOS 111-112), 652 (SEQ ID NOS 113-114), 653 (SEQ ID NOS115-116), 654 (SEQ ID NOS 117-118), 655 (SEQ ID NOS 119-120), 6565 (SEQID NOS 121-122), 51423 (SEQ ID NOS 123-124), and 27302 (SEQ ID NOS125-126)), Fgf (e.g. one or more of NCBI Gene IDs: 2252 (SEQ ID NOS127-128), 2255 (SEQ ID NOS 129-130), 9965 (SEQ ID NOS 131-132), 2249(SEQ ID NOS 133-134), 2248 (SEQ ID NOS 135-136), 2257 (SEQ ID NOS137-138), 8822 (SEQ ID NOS 139-140), 2251 (SEQ ID NOS 141-142), 27006(SEQ ID NOS 143-144), 2256 (SEQ ID NOS 145-146), 2247 (SEQ ID NOS147-148), 8074 (SEQ ID NOS 149-150), 2246 (SEQ ID NOS 151-152), 26291(SEQ ID NOS 153-154), 2253 (SEQ ID NOS 155-156), 2254 (SEQ ID NOS157-158), 2250 (SEQ ID NOS 159-160), 2258 (SEQ ID NOS 161-162), 8817(SEQ ID NOS 163-164), 26281 (SEQ ID NOS 165-166), and 2259 (SEQ ID NOS167-168)) Avtivin (e.g., NBCI Gene ID: 3624 (SEQ ID NOS 169-170)), andTGFβ (e.g. one or more of NCBI Gene IDs: 7040 (SEQ ID NOS 171-172), 7042(SEQ ID NOS 173-174), 7043 (SEQ ID NOS 175-176), and 7044 (SEQ ID NOS177-178)). In some embodiments, inhibiting the cell cycle can compriseculturing the cell in a media lacking one or more factors selected fromthe group consisting of: LIF; Bmp; Fgf; Activin; or TGFβ, e.g. lacking 1of the factors, 2 of the factors, 3 of the factors, 4 of the factors, 5of the factors, or more of the factors.

In some embodiments, contacting a cell with an agent can comprisecontacting the cell with one dose of the agent. In some embodiments,contacting a cell with an agent can comprise contacting the cell withrepeated doses of the agent. In some embodiments, contacting a cell withan agent can comprise maintaining a given concentration of the agent inthe cell's environment, e.g. in the culture media. In some embodiments,contacting a cell with an agent can comprise maintaining at least agiven minimum concentration of the agent in the cell's environment, e.g.in the culture media.

In some embodiments, the methods described herein can result in apopulation of cells comprising one or more terminally-differentiatedcell types, e.g. 1 terminally-differentiated cell type, 2terminally-differentiated cell types, 3 terminally-differentiated celltypes, 4 terminally-differentiated cell types, 5terminally-differentiated cell types, or more terminally-differentiatedcell types. When discussing a population of cells that results from themethods described herein, the resulting population can be the populationof cells existing at about 1 day to about 30 days after both steps (i)and (ii) have been performed.

In some embodiments, the methods described herein can result in apopulation of cells comprising no more than 2 terminally-differentiatedcell types, e.g. 1 terminally-differentiated cell type or 2terminally-differentiated cell types.

In some embodiments, the methods described herein can result in apopulation of cells of which at least about 50% of the cells areterminally-differentiated cell, e.g. about 50% or more of the cells areterminally-differentiated cells, about 60% or more of the cells areterminally-differentiated cells, about 70% or more of the cells areterminally-differentiated cells, about 80% or more of the cells areterminally-differentiated cells, about 90% or more of the cells areterminally-differentiated cells, about 95% or more of the cells areterminally-differentiated cells, or about 98% or more of the cells areterminally-differentiated cells. In some embodiments, the methodsdescribed herein can result in a population of cells of which at least50% of the cells are terminally-differentiated cell, e.g. 50% or more ofthe cells are terminally-differentiated cells, 60% or more of the cellsare terminally-differentiated cells, 70% or more of the cells areterminally-differentiated cells, 80% or more of the cells areterminally-differentiated cells, 90% or more of the cells areterminally-differentiated cells, 95% or more of the cells areterminally-differentiated cells, or 98% or more of the cells areterminally-differentiated cells.

Differentiated cells obtained in accordance with the methods describedherein can be used, e.g. for cell therapy, autologous cell therapy,transplantation, wound healing or repair, in vitro studies of cellfunction, cell growth, cell differentiation, and/or screens formodulators of cell function and behavior (e.g. therapeutics, drugcandidates, inhibitors or agonists of growth, function, anddifferentiation).

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, an “increase” is a statistically significant increasein such level.

-   As used herein, “contacting” refers to any suitable means for    delivering, or exposing, an agent to at least one cell. Exemplary    delivery methods include, but are not limited to, direct delivery to    cell culture medium, perfusion, injection, or other delivery method    well known to one skilled in the art.

The term “agent” refers generally to any entity which is normally notpresent or not present at the levels being administered to a cell,tissue or subject. An agent can be selected from a group including butnot limited to: polynucleotides; polypeptides; small molecules; andantibodies or antigen-binding fragments thereof. A polynucleotide can beRNA or DNA, and can be single or double stranded, and can be selectedfrom a group including, for example, nucleic acids and nucleic acidanalogues that encode a polypeptide. A polypeptide can be, but is notlimited to, a naturally-occurring polypeptide, a mutated polypeptide ora fragment thereof that retains the function of interest. Furtherexamples of agents include, but are not limited to a nucleic acidaptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), smallorganic or inorganic molecules; saccharide; oligosaccharides;polysaccharides; biological macromolecules, peptidomimetics; nucleicacid analogs and derivatives; extracts made from biological materialssuch as bacteria, plants, fungi, or mammalian cells or tissues andnaturally occurring or synthetic compositions. An agent can be appliedto the media, where it contacts the cell and induces its effects.Alternatively, an agent can be intracellular as a result of introductionof a nucleic acid sequence encoding the agent into the cell and itstranscription resulting in the production of the nucleic acid and/orprotein environmental stimuli within the cell. In some embodiments, theagent is any chemical, entity or moiety, including without limitationsynthetic and naturally-occurring non-proteinaceous entities. In certainembodiments the agent is a small molecule having a chemical moietyselected, for example, from unsubstituted or substituted alkyl,aromatic, or heterocyclyl moieties including macrolides, leptomycins andrelated natural products or analogues thereof. Agents can be known tohave a desired activity and/or property, or can be selected from alibrary of diverse compounds. As used herein, the term “small molecule”can refer to compounds that are “natural product-like,” however, theterm “small molecule” is not limited to “natural product-like”compounds. Rather, a small molecule is typically characterized in thatit contains several carbon-carbon bonds, and has a molecular weight morethan about 50, but less than about 5000 Daltons (5 kD). Preferably thesmall molecule has a molecular weight of less than 3 kD, still morepreferably less than 2 kD, and most preferably less than 1 kD. In somecases it is preferred that a small molecule have a molecular mass equalto or less than 700 Daltons.

As used herein, “ectopic” refers to a substance that is found in anunusual location and/or amount. An ectopic substance can be one that isnormally found in a given cell, but at a much lower amount and/or at adifferent time.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

The polypeptides of the present invention can be synthesized by usingwell known methods including recombinant methods and chemical synthesis.Recombinant methods of producing a polypeptide through the introductionof a vector including nucleic acid encoding the polypeptide into asuitable host cell are well known in the art, e.g., as described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed, Vols 1to 8, Cold Spring Harbor, N.Y. (1989); M. W. Pennington and B. M. Dunn,Methods in Molecular Biology: Peptide Synthesis Protocols, Vol 35,Humana Press, Totawa, N.J. (1994), contents of both of which are hereinincorporated by reference. Peptides can also be chemically synthesizedusing methods well known in the art. See for example, Merrifield et al.,J. Am. Chem. Soc. 85:2149 (1964); Bodanszky, M., Principles of PeptideSynthesis, Springer-Verlag, New York, N.Y. (1984); Kimmerlin, T. andSeebach, D. J. Pept. Res. 65:229-260 (2005); Nilsson et al., Annu. Rev.Biophys. Biomol. Struct. (2005) 34:91-118; W. C. Chan and P. D. White(Eds.) Fmoc Solid Phase Peptide Synthesis: A Practical Approach, OxfordUniversity Press, Cary, N.C. (2000); N. L. Benoiton, Chemistry ofPeptide Synthesis, CRC Press, Boca Raton, Fla. (2005); J. Jones, AminoAcid and Peptide Synthesis, 2^(nd) Ed, Oxford University Press, Cary,N.C. (2002); and P. Lloyd-Williams, F. Albericio, and E. Giralt,Chemical Approaches to the synthesis of peptides and proteins, CRCPress, Boca Raton, Fla. (1997), contents of all of which are hereinincorporated by reference. Peptide derivatives can also be prepared asdescribed in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, andU.S. Pat. App. Pub. No. 2009/0263843, contents of all which are hereinincorporated by reference.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

In some embodiments, the technology described herein relates to anucleic acid encoding a polypeptide as described herein. As used herein,the term “nucleic acid” or “nucleic acid sequence” refers to anymolecule, preferably a polymeric molecule, incorporating units ofribonucleic acid, deoxyribonucleic acid or an analog thereof. Thenucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one strand nucleic acid of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the template nucleic acid is DNA. In another aspect, thetemplate is RNA. Suitable nucleic acid molecules include DNA, includinggenomic DNA or cDNA. Other suitable nucleic acid molecules include RNA,including mRNA. The nucleic acid molecule can be naturally occurring, asin genomic DNA, or it may be synthetic, i.e., prepared based upon humanaction, or may be a combination of the two. The nucleic acid moleculecan also have certain modification(s) such as 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA), cholesteroladdition, and phosphorothioate backbone as described in US PatentApplication 20070213292; and certain ribonucleoside that are linkedbetween the 2′-oxygen and the 4′-carbon atoms with a methylene unit asdescribed in U.S. Pat. No. 6,268,490, wherein both patent and patentapplication are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid encoding a polypeptide as describedherein is comprised by a vector. In some of the aspects describedherein, a nucleic acid sequence encoding a polypeptide as describedherein is operably linked to a vector. The term “vector”, as usedherein, refers to a nucleic acid construct designed for delivery to ahost cell or for transfer between different host cells. As used herein,a vector can be viral or non-viral. The term “vector” encompasses anygenetic element that is capable of replication when associated with theproper control elements and that can transfer gene sequences to cells. Avector can include, but is not limited to, a cloning vector, anexpression vector, a plasmid, phage, transposon, cosmid, chromosome,virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain a nucleic acid encoding a polypeptide as described herein inplace of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring nucleic acids into cells eitherin vitro or in vivo. Numerous forms of viral vectors are known in theart.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra chromosomal DNAthereby eliminating potential effects of chromosomal integration.

The term “progenitor cell” is used herein to refers to cells that have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate.

The term “stem cell” as used herein, refers to an undifferentiated cellwhich is capable of proliferation and giving rise to more progenitorcells having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated, or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers to a subset ofprogenitors that have the capacity or potential, under particularcircumstances, to differentiate to a more specialized or differentiatedphenotype, and which retains the capacity, under certain circumstances,to proliferate without substantially differentiating. In one embodiment,the term stem cell refers generally to a naturally occurring mother cellwhose descendants (progeny) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a process typically occurring through manycell divisions. A differentiated cell may derive from a multipotent cellwhich itself is derived from a multipotent cell, and so on. While eachof these multipotent cells may be considered stem cells, the range ofcell types each can give rise to may vary considerably. Somedifferentiated cells also have the capacity to give rise to cells ofgreater developmental potential. Such capacity may be natural or may beinduced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stem-ness.” Self-renewal is the other classical part ofthe stem cell definition, and it is essential as used in this document.In theory, self-renewal can occur by either of two major mechanisms.Stem cells may divide asymmetrically, with one daughter retaining thestem state and the other daughter expressing some distinct otherspecific function and phenotype. Alternatively, some of the stem cellsin a population can divide symmetrically into two stems, thusmaintaining some stem cells in the population as a whole, while othercells in the population give rise to differentiated progeny only.

The term “embryonic stem cell” is used to refer to the pluripotent stemcells of the inner cell mass of the embryonic blastocyst (see U.S. Pat.Nos. 5,843,780, 6,200,806, which are incorporated herein by reference).Such cells can similarly be obtained from the inner cell mass ofblastocysts derived from somatic cell nuclear transfer (see, forexample, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which areincorporated herein by reference). The distinguishing characteristics ofan embryonic stem cell define an embryonic stem cell phenotype.Accordingly, a cell has the phenotype of an embryonic stem cell if itpossesses one or more of the unique characteristics of an embryonic stemcell such that that cell can be distinguished from other cells.Exemplary distinguishing embryonic stem cell characteristics include,without limitation, gene expression profile, proliferative capacity,differentiation capacity, karyotype, responsiveness to particularculture conditions, and the like.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. As indicated above, stemcells have been found resident in virtually every tissue. Accordingly,the technology described herein appreciates that stem cell populationscan be isolated from virtually any animal tissue. As used herein, theterm “adult cell” refers to a cell found throughout the body afterembryonic development.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and refers to a pluripotent cell artificiallyderived (e.g., induced by complete or partial reversal) from adifferentiated somatic cell (i.e. from a non-pluripotent cell). Apluripotent cell can differentiate to cells of all three developmentalgerm layers.

The term “derived from” used in the context of a cell derived fromanother cell means that a cell has stemmed from (e.g. changed from orwas produced by) a cell which is a different cell type. In someinstances, for example, a cell derived from an iPS cell refers to a cellwhich has differentiated from an iPS cell. Alternatively, a cell can beconverted from one cell type to a different cell type by a processreferred to as transdifferention or direct reprogramming. Alternatively,in the terms of iPS cells, a cell (e.g. an iPS cell) can be derived froma differentiated cell by a process referred to in the art asdedifferentiation or reprogramming.

The term “pluripotent” as used herein refers to a cell that can giverise to any type of cell in the body except germ line cells. The term“pluripotency” or a “pluripotent state” as used herein refers to a cellwith the ability to differentiate into all three embryonic germ layers:endoderm (gut tissue), mesoderm (including blood, muscle, and vessels),and ectoderm (such as skin and nerve), and typically has the potentialto divide in vitro for a long period of time, e.g., greater than oneyear or more than 30 passages. Pluripotency is also evidenced by theexpression of embryonic stem (ES) cell markers, although the preferredtest for pluripotency is the demonstration of the capacity todifferentiate into cells of all three germ layers, as detected using,for example, a nude mouse teratoma formation assay. iPS cells arepluripotent cells. Pluripotent cells undergo further differentiationinto multipotent cells that are committed to give rise to cells thathave a particular function. For example, multipotent cardiovascular stemcells give rise to the cells of the heart, including cardiomyocytes, aswell as other cells involved in the vasculature of the heart.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

The term “lineages” as used herein refers to a term to describe cellswith a common ancestry, for example cells that are derived from the samecardiovascular stem cell or other stem cell, or cells with a commondevelopmental fate. By way of an example only, when referring to a cellthat is of endoderm origin or is “endodermal linage,” this means thecell was derived from an endodermal cell and can differentiate along theendodermal lineage restricted pathways, such as one or moredevelopmental lineage pathways which give rise to definitive endodermcells, which in turn can differentiate into liver cells, thymus,pancreas, lung and intestine.

In the context of cell ontogeny, the term “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an atrial precursor), and then to anend-stage differentiated cell, such as atrial cardiomyocytes or smoothmuscle cells, which play a characteristic role in a certain tissue type,and may or may not retain the capacity to proliferate further. The term“differentiated cell” refers to any primary cell that is not, in itsnative form, pluripotent as that term is defined herein. The term a“differentiated cell” also encompasses cells that are partiallydifferentiated, such as multipotent cells, or cells that are stablenon-pluripotent partially reprogrammed cells. In some embodiments, adifferentiated cell is a cell that is a stable intermediate cell, suchas a non-pluripotent partially reprogrammed cell. It should be notedthat placing many primary cells in culture can lead to some loss offully differentiated characteristics. However, simply culturing suchprimary cells, e.g., after removal or isolateion from a tissue ororganism does not render these cells non-differentated cells (e.g.undifferentiated cells) or pluripotent cells. The transition of adifferentiated cell (including stable non-pluripotent partiallyreprogrammed cell intermediates) to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture.

The term “differentiation” as referred to herein refers to the processwhereby a cell moves further down the developmental pathway and beginsexpressing markers and phenotypic characteristics known to be associatedwith a cell that are more specialized and closer to becoming terminallydifferentiated cells. The pathway along which cells progress from a lesscommitted cell to a cell that is increasingly committed to a particularcell type, and eventually to a terminally differentiated cell isreferred to as progressive differentiation or progressive commitment.Cell which are more specialized (e.g., have begun to progress along apath of progressive differentiation) but not yet terminallydifferentiated are referred to as partially differentiated.Differentiation is a developmental process whereby cells assume a morespecialized phenotype, e.g., acquire one or more characteristics orfunctions distinct from other cell types. In some cases, thedifferentiated phenotype refers to a cell phenotype that is at themature endpoint in some developmental pathway (a so called terminallydifferentiated cell). In many, but not all tissues, the process ofdifferentiation is coupled with exit from the cell cycle. In thesecases, the terminally differentiated cells lose or greatly restricttheir capacity to proliferate. However, in the context of thisspecification, the terms “differentiation” or “differentiated” refer tocells that are more specialized in their fate or function than at onetime in their development. For example in the context of thisapplication, a differentiated cell includes a cardiomyocyte which hasdifferentiated from cardiovascular progenitor cell, where suchcardiovascular progenitor cell can in some instances be derived from thedifferentiation of an ES cell, or alternatively from the differentiationof an induced pluripotent stem (iPS) cell, or in some embodiments from ahuman ES cell line. A cell that is “differentiated” relative to aprogenitor cell has one or more phenotypic differences relative to thatprogenitor cell and characteristic of a more mature or specialized celltype. Phenotypic differences include, but are not limited to morphologicdifferences and differences in gene expression and biological activity,including not only the presence or absence of an expressed marker, butalso differences in the amount of a marker and differences in theco-expression patterns of a set of markers.

The term “contacting” or “contact” as used herein in connection withcontacting a cell with an agent as described herein, includes subjectingthe cell to a culture medium which comprises that agent.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. A method of differentiating a stem cell, the method comprising:

-   -   i) contacting the stem cell with one or more ectopic        differentiation factors; and    -   ii) inhibiting the cell cycle of the stem cell;    -   wherein steps i) and ii) occur within 15 days of each other.        2. The method of paragraph 1, wherein steps i) and ii) occur        within 14 days of each other.        3. The method of paragraph 1, wherein steps i) and ii) occur        within 13 days of each other.        4. The method of paragraph 1, wherein steps i) and ii) occur        within 12 days of each other.        5. The method of paragraph 1, wherein steps i) and ii) occur        within 11 days of each other.        6. The method of paragraph 1, wherein steps i) and ii) occur        within 10 days of each other.        7. The method of paragraph 1, wherein steps i) and ii) occur        within 9 days of each other.        8. The method of paragraph 1, wherein steps i) and ii) occur        within 8 days of each other.        9. The method of paragraph 1, wherein steps i) and ii) occur        within 7 days of each other.        10. The method of paragraph 1, wherein steps i) and ii) occur        within 6 days of each other.        11. The method of paragraph 1, wherein steps i) and ii) occur        within 5 days of each other.        12. The method of paragraph 1, wherein steps i) and ii) occur        within 4 days of each other.        13. The method of paragraph 1, wherein steps i) and ii) occur        within 3 days of each other.        14. The method of paragraph 1, wherein steps i) and ii) occur        within 2 days of each other.        15. The method of paragraph 1, wherein steps i) and ii) occur        within 24 hours of each other.        16. The method of paragraph 1, wherein steps i) and ii) occur        simultaneously.        17. The method of any of paragraphs 1-16, wherein the        differentiation factor is a terminal transcription factor.        18. The method of paragraph 17, wherein the terminal        transcription factor is selected from Table 1.        19. The method of any of paragraphs 17-18, wherein the stem cell        is to be differentiated to a skeletal muscle phenotype and is        contacted with the terminal transcription factor MyoD.        20. The method of any of paragraphs 17-18, wherein the stem cell        is to be differentiated to a spinal motor neuron phenotype and        is contacted with a terminal transcription factor selected from        the group consisting of: Ngn2; Isl1; and Lhx3.        21. The method of any of paragraphs 17-18, wherein the stem cell        is to be differentiated to a spinal motor neuron phenotype and        is contacted with the terminal transcription factors Ngn2; Isl1;        and Lhx3.        22. The method of any of paragraphs 17-18, wherein the stem cell        is to be differentiated to a cardiomyocyte phenotype and is        contacted with the terminal transcription factor Gata5.        23. The method of any of paragraphs 17-18, wherein the stem cell        is to be differentiated to a hepatocyte or hepatoblast phenotype        and is contacted with the terminal transcription factor Hnf4α.        24. The method of any of paragraphs 1-23, wherein the cell cycle        is inhibited by one or more of the following:    -   reducing or removing growth factors; reducing serum levels;        reducing serum levels below 5%; contacting the cell with a PI3K        inhibitor; contacting the cell with an E2F family transcription        factor inhibitor; contacting the cell with a Myc inhibitor;        contacting the cell with a MAPK inhibitor; contacting the cell        with a MEK1/2 inhibitor; contacting the cell with a CDK        inhibitor; contacting the cell with an Id inhibitor; contacting        the cell with a Rb agonist; contacting the cell with a Ink        family agonist; contacting the cell with a Cip/Kip family        agonist; and culturing the cell in a media lacking a factor        selected from the group consisting of:        -   LIF; Bmp; Fgf; Activin; or TGFβ.            25. The method of paragraph 24, wherein the cell cycle is            inhibited by reducing or removing growth factors.            26. The method of paragraph 24, wherein the cell cycle is            inhibited by culturing the cell in a media lacking a factor            selected from the group consisting of:    -   LIF; Bmp; Fgf; Activin; or TGFβ.        27. The method of paragraph 26, wherein the cell cycle is        inhibited by culturing the cell in a media lacking LIF.        28. The method of paragraph 24, wherein the PI3K inhibitor is        LY294002.        29. The method of paragraph 24, wherein the E2F transcription        factor inhibitor is HLM006474.        30. The method of paragraph 24, wherein the Myc inhibitor is JQ1        or 10058-F4.        31. The method of paragraph 24, wherein the MAPK inhibitor is        PD98059.        32. The method of paragraph 24, wherein the CDK inhibitor is a        CDK4 or CDK2 inhibitor.        33. The method of paragraph 24, wherein the CDK inhibitor is        p16, p15, p18, or p19.        34. The method of paragraph 24, wherein the CDK inhibitor is        p21, p27, or p57.        35. The method of any of paragraphs 1-34, wherein the stem cell        is an embryonic stem cell.        36. The method of any of paragraphs 1-35, wherein steps i)        and ii) result in a population of cells comprising one or more        terminally-differentiated cell types.        37. The method of any of paragraphs 1-35, wherein steps i)        and ii) result in a population of cells comprising no more than        2 terminally-differentiated cell types.        38. The method of any of paragraphs 1-35, wherein steps i)        and ii) result in a population of cells of which at least 50%        are terminally-differentiated cells.        39. The method of any of paragraphs 1-35, wherein steps i)        and ii) result in a population of cells of which at least 60%        are terminally-differentiated cells.        40. The method of any of paragraphs 1-35, wherein steps i)        and ii) result in a population of cells of which at least 70%        are terminally-differentiated cells.        41. The method of any of paragraphs 1-35, wherein steps i)        and ii) result in a population of cells of which at least 80%        are terminally-differentiated cells.

Examples Example 1: Molecular Ties Between the Cell Cycle andDifferentiation in Embryonic Stem Cells

Attainment of the differentiated state during the final stages ofsomatic cell differentiation is closely tied to cell cycle progression.Much less is known about the role of the cell cycle at very early stagesof embryonic development. It is demonstrated herein that molecularpathways involving the cell cycle can be engineered to strongly affectembryonic stem cell differentiation at early stages in vitro. Strategiesbased on perturbing these pathways can shorten the rate and simplify thelineage path of ES differentiation. These results make it likely thatpathways involving cell proliferation intersect at various points withpathways that regulate cell lineages in embryos and demonstrate thatthis knowledge can be used profitably to guide the path andeffectiveness of cell differentiation of pluripotent cells.

As cells differentiate during embryonic development, they progressthrough a stereotypical sequence of events, starting from highly potentembryonic precursors to germ layer intermediates, then tolineage-restricted progenitors, and finally, to terminallydifferentiated cell types. Any of these stages may consist of furtherstates of differentiation and may be difficult to recognize. Most of ourknowledge about the differentiation process comes from studies in thelatter stages of differentiation (i.e. terminal model systems), wherecells are one step away from their final fate and are usually restrictedto differentiate to one type of cell. Less is known about what happensduring early embryonic stages, where the differentiation process is justbeginning and many alternative pathways of differentiation may still beavailable.

In terminal somatic cell culture models, inhibition of the cell cycle isalmost always a requisite for differentiation. Forced inhibition of thecell cycle very often induces terminal differentiation and vice versa(1-3). The molecular pathways that couple the cell cycle todifferentiation involve molecules of the G1/S transition includinggrowth factors, downstream signaling pathways, Myc, the Rb/E2F pathway,and the CDK inhibitors (e.g. p21). The role of G1 length on embryonicstem cell self-renewal was investigated and it was found that incontrast to the terminal stages it did not accelerate the loss ofpluripotency or facilitate differentiation (4). Described herein is thestate of the cell cycle molecular network in the ES cell system and howthe cell cycle may be re-coupled to differentiation to re-direct lineagepathways, e.g., for practical benefit.

Results

In terminally differentiated cells the cell cycle and differentiationare linked together through a molecular network rooted in the G1/Stransition. A wiring diagram summarizing such a network is shown in FIG.1A, with explanations and justifications provided in FIGS. 7A-7C, whichwas constructed from known or postulated relationships in normal somaticcycling cells, and interactions between the cell cycle machinery andterminal transcription factors, such as MyoD.

During the process of differentiation, the network changes. For culturedcells at the terminal stage of differentiation, this involves an exitfrom the cell cycle, activation of terminal transcription factors, and ashift towards insulin signaling away from other growth factors forsurvival and growth. These changes to the network are shown in FIG. 1B.At the other end of the differentiation process are embryonic stemcells, which have a number of unique features. They can be maintained inan undifferentiated state with the combination of Leukemia InhibitoryFactor (LIF) and high amounts of serum, or LIF and Bmp4, as shown inFIG. 1C. The actual differentiation process from ES cells to terminallydifferentiated cells spans at least three states in a defined order (ESto somatic cycling cells to terminal differentiation), but may passthrough other intermediate stages of differentiation, expressing genesand behavior different from terminal cells and pluripotent stem cells;little is known concerning the cell cycle and their state ofdifferentiation in these largely uncharacterized cell cycle states (FIG.1D).

Guided Differentiation and Cell Cycle Manipulation of ES Cells.

To probe the effect of the cell cycle on differentiation an ES cell linewas used that constitutively over-expressed the transcription factorMyoD driven off an EF1alpha promoter (5) activated by tamoxifen-inducedCre recombination. The use of this cell line facilitated the analysis bychanneling differentiation away from a diverse collection of phenotypesinto a more uniform population of cells expressing muscle genes, such asmyosin heavy chain (MHC). The first cell cycle manipulation was growthfactor or serum withdrawal. Using the cell line that continuouslyexpressed MyoD, LIF was removed at what is referred to herein as zerotime to initiate differentiation and serum was reduced at various timesthereafter from the standard 15% serum to 2% with additional insulin (10μg/ml). As shown in FIG. 2A, in the continuous presence of 15% serum,MHC is completely suppressed, despite constitutive MyoD overexpression.When serum is reduced one day after MyoD induction, MHC begins toaccumulate four days later. By day 12, 20-30 percent of cells expressMHC and show characteristic morphology of mature skeletal muscle fibersincluding elongation, increase in volume, and significantmulti-nucleation (FIG. 8). If serum removal is delayed relative to LIFremoval, the cells still begin to express MHC with a 2 to 4 day delayafter serum reduction. Thus serum reduction strongly potentiatesterminal muscle differentiation in a very short time under theconditions studied.

From the cell cycle summary in FIG. 1A, MyoD activation and hence muscledifferentiation from ES cells should be blocked by either the action ofLIF, which activates Myc, or Bmp4, which promotes Id protein familyexpression. However any implication of growth factor effects through themanipulation of serum can be fraught with the inconsistencies andcomplexities of serum. To avoid these problems more defined conditionswere examined with two types of basal insulin-containing media, N2B27and DMEM plus 20% Knock-out Serum Replacement (KOSR), neither of whichcontains growth factors. Use of both media in ES cells led to activationof MyoD and terminal myogenesis similar to the 2% low serum media, withsome improvement (FIG. 2B; assessed on Day 4). The N2B27 media producedapproximately 38.5% MHC+nuclei while 20% KOSR produced approximately19.6%. As expected when LIF or Bmp4 was added back to N2B27differentiation was blocked (0%; FIG. 2B). These results confirm theexpectation that the reduction of LIF and BMP in the setting of no othergrowth factors, produces highly effective conditions for ESdifferentiation.

The lineage from ES cells to terminal differentiation first involves theloss of pluripotency factors, followed by passage through intermediatecell types, identifiable by expression of specific transcriptionfactors. It was found that the decline in Oct4 and Nanog mRNA levelsinduced by LIF removal was completely unaffected by serum reduction.This is similar to results showing that extension of G1 had no effect onNanog levels (4).

By contrast, beyond the loss of pluripotency factors there is a dramaticeffect of serum removal on the differentiation cascade toward muscle.From studies in embryos, there is a prescribed sequence of steps insetting up the myogenic lineage involving the specification of themesoderm, the sub-specification of the myotome, and the steps leading toovert cell differentiation (6-7). When the mRNA levels of genes withinthis hierarchy were examined using the above protocol of serumreduction, it was found that Pax3, which is expressed in thedermomyotome, rose dramatically (to a peak of ˜50 fold) and veryprematurely within 2 days of serum reduction. The pre-myogenichomeodomain factors Six1 (8) and Six4 (9), which are normally upstreamof Pax3, are not affected or modestly suppressed, as was the case forthe paired-box domain protein Pax7, which is expressed in thedermomyotome and somites during embryogenesis (10). There are smalleffects of serum reduction on the myogenic regulatory factor (MRF)genes, like Myf5, MRF4 and endogenous MyoD, but there is a massive (300fold) upregulation of myogenin (MyoG), which plays a key role in verylate-stage skeletal myogenesis during the period of days 3 to 7 (11).Other muscle lineage markers also respond rapidly to serum withdrawal inthe presence of MyoD, indicating that the entire suite of terminalmuscle lineage is induced very prematurely. Seven of these—desmin,skeletal muscle actin, troponin, myosin light chain, tropomyosin, themyoblast fusion regulator Dyrk1b (12), and titin—are shown in FIG. 9.The dramatic overexpression of Pax3 and MyoG depend on theover-expressed exogenous MyoD, as without the induction of MyoD, theirexpression is lower. These results document the extraordinarily rapidproduction of some downstream muscle differentiation factors anddefinitive muscle proteins in the setting of growth factor or serumwithdrawal.

Promoting Differentiation by Perturbing Intracellular Pathways.

Based on suggestions from the pathway diagrams in FIGS. 1A-1D, a fewcritical components of cell cycle control were focused on and theireffects on the two markers strongly perturbed by serum withdrawal, Pax3and MyoG, were measured. Perturbations were made both under high and lowserum conditions and were extended throughout the time course ofdifferentiation. LY294002 is a potent broad inhibitor ofphosphoinositide-3-kinases (PI3Ks), and when applied continuously to EScells over 7 days induced a significant 2.7 fold increase in Pax3 mRNAexpression (FIG. 4). This increase was observed both in high serum andlow serum media. However, continuing treatment with LY294002 led to celldeath and no expression of myogenin was observed (FIG. 4, bottompanels). A similar situation was observed with HLM006474, which broadlyinhibits E2F family transcription factors in their interaction with DPproteins. Myc drives cell cycle progression and growth. Its activity canbe inhibited by two compounds: JQ1, a newly-identified compound thatspecifically inhibits bromodomains but subsequently results in Mycdownregulation, and 10058-F4, an inhibitor that blocks the dimerizationof Myc-Max complexes. Both are indirect inhibitors of Myc activity; asyet there are no direct pharmacologic inhibitors of the Myc protein. Theeffects of JQ1 were similar to LY294002 and HLM00647: an increase inPax3 early expression, but later suppression of MyoG expression. Out ofall drugs the Myc-bromodomain inhibitor JQ1 had the largest effect ininducing Pax3, whereas the Myc-Max dimerization inhibitor 10058-F4 hadsuppressive effects on both Pax3 and MyoG.

The effects of inhibiting cyclin-dependent kinases (kinases that aremore centrally involved in cell cycle control) and MAP kinase, whichvery often is involved in cell cycle regulation, were also examinedRoscovitine is a broad CDK inhibitor that blocks a number of familymembers, including CDK1, CDK2, and CDK5. After continuous treatmentthroughout the 7 days of differentiation in the time course, roscovitinehad no effect on Pax3 expression (FIG. 4). It also had little effect onthe later expression of MyoG, either under high or low serum conditions.This lack of effect on Pax3 and MyoG was also observed for the morespecific CDK4 inhibitor PD0332991. However, the MAPK inhibitor PD98059,which blocks MEK1/2, had no effect on Pax3 and induced MyoG only underlow serum conditions (FIG. 4 bottom). The protein CDK inhibitor p21,which is much more specific than roscovitine, blocks CDK2 and preventsentry into S-phase. In ES cells p21 is expressed at low levels, butgradually increases during the course of ES cell differentiation (13).An mES cell line that constitutively overexpresses the p21 proteinbicistronically with a mCherry tag (fused to the C-terminus with a 2Apeptide) was developed. This line exhibits an elongated G1 and can bepropagated in standard LIF+serum media. When induced to differentiate bythe removal of LIF, this line upregulated MyoG under low serum media,but had no effect on Pax3, a behavior similar to the MAPK inhibitor.Thus, within the set of cell cycle inhibitors examined, stage-specificand condition-specific effects on gene expression were observed.

Induction of Unguided Differentiation.

Although forced expression of MyoD nicely served to focusdifferentiation into the skeletal muscle cell lineage, it was alsodesired to examine what happens in ES cells that are not guided in theirdifferentiation path by MyoD. When LIF is removed in ES cells withoutMyoD, there is differentiation into a heterogeneous mixture of celltypes. Under standard culture conditions of 15% serum, which promotesexpansion, ES cells deprived of LIF normally differentiate first intogeneral mesodermal, endodermal, and ectodermal tissues, and then laterinto a heterogeneous mixture of terminal cell types (7, 14). The effectsof serum withdrawal on this system were examined. When high and lowserum time courses in ES cells differentiating without exogenous MyoDwere compared over a period of 7 days there was premature expression ofgenes that are normally associated with multiple cell lineages (FIG. 5).Low serum induced the expression of many lineage-specific factors. Forexample, an increase in the neural marker Delta (Dll1) was observed. Thecardiac muscle factors Sox6, Smyd1, GATA4, GATA6 all increased, as wellas the neural/muscle transcription factor Mef2c. For adipose genes, avery large increase in PPARγ expression (>400 fold) was detected. Theearly endoderm genes Sox17 and Nkx2.2 also were elevated in low serumcompared to high serum. Similarly, increases in Runx2 (osteoblastdifferentiation), Mitf (melanocyte), and Sox9 (chondrocytedifferentiation) were also observed. For hematopoietic factors,increases in the erythrocyte factor GATA1 (FIG. 5) and the progenitorfactor GATA2 (FIG. 10) were observed. A full list of factors that wereprofiled and their time course data is available in FIG. 10.

The early upregulation of such a large number of somatic lineage factorsindicates that growth factor/serum reduction is permissive for a widevariety of differentiated gene expression. Many of the upregulatedfactors have been reported to function in terminal differentiation.Perhaps most interesting is the failure to express many of the markersof the early lineages. As seen in the MyoD-guided system, only Pax3 andMyoG were significantly activated, but not other factors in the musclelineage hierarchy. In the unguided system, in addition to the terminalfactors that were upregulated, there were numerous intermediate lineagefactors that were not (e.g. Pax6, C/EBPα, C/EBPβ, Pdx1, Cdx2, etc.)(FIG.10 and Table 3).

Discussion

Our understanding of cell differentiation comes mainly from twodifferent sources: studies of cell culture systems and studies ofembryonic systems. Although the embryo remains the gold standard for thefunctional process of embryogenesis, there is today a strong incentiveto understand alternative in vitro pathways that can be exploited fortherapeutic purposes. Furthermore there is no reason why we shouldconsider embryonic lineages as mechanistically the most informative.Embryos have to accomplish feats other than differentiation, such asmorphogenesis and cell proliferation, and many intermediate behaviors ofcells may reflect those roles.

Much ingenuity and decades of effort has resulted in the discovery ofways to manipulate cells isolated and cultured from various tissues sothat they can differentiate into one or a very few cell types. It is nowrecognized that these processes take cells from an already determinedstate and drive them to a state of clear expression of specific markers,rather than starting from a very early precursor state. Suchmanipulations can drive presumptive myoblasts to muscle, neuroblasts toneurons, fibroblasts to adipocytes, etc. A very different source ofcells are pluripotent ES cells of the mouse and now of human. Thesecells start at an earlier state and can be driven to differentiateeither by re-creating some early embryonic state through embryoid bodiesor by going through a series of steps in culture, thought to parallelthe various intermediate states of differentiation found in the embryoitself. As work in stem cells and ES cells in particular exploded in thelast few years there has been a serious effort to identify and recreatein culture the series of signals that drive the pluripotent state to thedifferentiated state. In the embryo, these include the addition andremoval of factors like Wnts, Nodals, BMPs, EGFs, etc. There is both apractical side to this endeavor: to either generate differentiated cellsthat can generate replacement tissues and organs or to find ways tostimulate the body's regenerative potential to repair worn or diseasedcells.

There has also been a long standing interest in understanding how thecell cycle could further the process of differentiation Inhibition ofcell proliferation in G1 is almost always accompanied by celldifferentiation. The work described herein was designed to determinewhether this effect of cell cycle inhibition can be observed early inthe differentiation process and whether the paths taken are the same asseen in the absence of cell cycle inhibition.

Described herein are cell cycle perturbations, starting with a reductionor removal of growth factors/serum, change the timing and the course ofdifferentiation to muscle in a model that involves the continuousexpression of MyoD. Notably none of the perturbations had an effect onexit from the ES cell state as reflected in the loss of Oct4 and Nanog.Surprisingly, this progression to the differentiated state seemed not toseem to follow the normal sequence of gene expressions seen in ES cellsin culture or by embryonic lineages in the embryo. More than one path todifferentiation was found, as described herein.

Described herein are heuristic descriptions of somatic cycling cells,terminally differentiating cells, and embryonic stem cells (FIGS.1A-1D). Terminally differentiated cells typically maintain their size orgrow slowly, stimulated commonly by the insulin pathway. In this cellcycle state the drivers of the cell cycle are inhibited and theexpression of cell cycle inhibitors are stimulated, leading to theexpression of terminally differentiated genes like MyoD, neurogenin,etc. The proliferative state that preceded terminal differentiation isreduced in the expression of CDK inhibitors and particularly in theactivity of Rb. In this case MyoD activity is also reduced. Lastly, thepluripotent embryonic state has new extracellular players: LIF, serumgrowth factors, and BMP4. In this state, there is more completesuppression of Rb and MyoD through the activation of proliferativesignals in the cell cycle and the downstream suppression ofanti-proliferative factors such as CDK inhibitors.

The results described herein indicate that in the MyoD-guided systemthere are minimally 3 states and two transitions (FIG. 6, top box). Thefirst stable state is pluripotency. When the cell cycle is suppressedthere is a rapid transition to an intermediate somatic state, whichcorrelates with an upregulation in Pax3. Manipulations that promote thistransition also promote Pax3 upregulation. For example, this transitioncould be facilitated by growth factor/serum withdrawal, LY294002 (PI3Kinhibition), HLM006474 (E2F inhibition), and JQ1 (Bromodomain/Mycinhibition). Subsequently there is a second transition to the terminallydifferentiating state, which correlates with upregulation of MyoG.Manipulations that promote this transition also promote MyoG. MyoG couldbe facilitated by PD98059 (MEK1/2 inhibitor), and p21 (CDK inhibitor)when combined with low serum, but not with roscovitine (broad CDK familyinhibitor) or PD0332991 (CDK4 inhibitor).

Though this scheme is likely to be a simplification for any lineage andmay differ in different lineages, it nevertheless helped to make senseof a number of observations. The removal of extracellular factors in theform of LIF and Bmp4, and the replacement of serum with insulin leads toactivation of MyoD and full induction of terminal myogenesis. Whenapplied early, this leads to a very direct form of ES-to-terminaldifferentiation, and Pax3 and MyoG upregulation is observed. PI3Kinhibition is predicted to activate MyoD by removing the stimulus toMyc, but is also expected to be inhibitory on the last steps of terminaldifferentiation as it becomes necessary for metabolic cell growth of thefinal differentiated cell. Accordingly, LY294002 upregulated Pax3 butpromoted poor survival, which could have suppressed MyoG expression.This suggests that the step of MyoG upregulation corresponds to whathappens in the terminal phase in this model, which is consistent withwhat is known about myogenin's role from terminal models. From theheuristic description, it is also expected that Myc or E2F suppressionwould help activate MyoD. JQ1 and HLM006474 both induced Pax3, which isconsistent with this prediction. However, it was also noticed that theysuppressed MyoG expression.

The cell cycle schemes also correctly predict that some CDK inhibitorsand the MAPK inhibitor would have little effect on Pax3 expression bythemselves, but would facilitate MyoG expression. This is due to thefact that in ES cells Myc and Id proteins are highly expressed and canindependently repress the expression of endogenous MyoD and othermyogenic regulatory factors outside of CDK activity (FIG. 1C). As EScells differentiate, they transition to the somatic cell cycling stateand then to the terminally differentiated model (FIG. 1D). Throughoutthis transition Myc and Id activity decline until CDK activity is thepredominant factor blocking differentiation. Hence at the early stage ofPax3 activation CDK inhibition is not expected to have a significanteffect, whereas at the late stage of MyoG activation the expectation isthat it will. Moreover, CDK4 activity is suppressed in ES cells, soadditional inhibition of their activity by PD0332991 should reveal noeffect on Pax3. MAPK (MEK1/2) activity is also suppressed in ES cells,but is upregulated during differentiation. Thus its inhibition shouldstimulate MyoG induction, but not Pax3 induction.

It is described herein that the CDK inhibitors and the MAPK inhibitorpromote differentiation only under low serum conditions, as high levelsof growth factor/serum conditions are expected to induce higher levelsof Myc and Id expression. It is further described herein that differentCDK inhibitors can have different effects. The p21 protein is highlyeffective at promoting MyoG expression, but not roscovitine orPD0332991. Without wishing to be bound by theory, this may have to dowith the differing specificities of the kinase inhibitors used.

It is specifically contemplated herein that the methods described hereincan permit the differentiation of other cell types, e.g., with otherterminal factors in place of MyoD. Indeed, many terminal cell systemscouple cell cycle inhibition with differentiation (1), and many potenttranscription factors interact with cell cycle components in wayssimilar to MyoD, including Ngn2, Pdx1, Smad3, Mitf, Runx2, PU.1, Hnf4α,and C/EBPβ (17-24). Expression of several genes associated with diversedifferentiated cell types could be stimulated by cell cycle inhibitionin a heterogeneous differentiation system where MyoD was not expressed(FIG. 5).

The forced silencing of proliferative pathways and the resulting rapiddifferentiation in vitro described herein is a useful strategy togenerate terminal cell types as compared to trying to recapitulateembryonic differentiation pathways, which often takes weeks (FIG. 6,bottom box)(26-31).

Materials and Methods

ES culture and differentiation. The ES cells contain MyoD (andassociated puromycin resistance marker) expressed from a EF1alphapromoter (5). MyoD could be expressed once a loxP segment insertedbetween the promoter and transgene was excised by a cre recombinasefused to the estrogen receptor. ES cells were cultured in LIF andstandard conditions containing 15% FBS, non-essential amino acids,L-glutamine, penicillin/streptomycin, and beta-mercaptoethanol. Toinduce MyoD, cells were treated for 24 hrs with 1 μM 4OHT (Sigma) in ESmedia. Reduced serum media consisted of DMEM and 2% horse serum(Invitrogen) plus 10 μg/ml insulin (to maintain cell survival)(Sigma)with sodium pyruvate and penicillin/streptomycin. Duringdifferentiation, cells were treated with 1 μg/ml puromycin continuouslyto select for cells which maintained MyoD expression. N2B27components/supplements and Knock-out serum replacement were purchasedfrom Invitrogen. LIF was used at a 1000 U/ml and Bmp4 at 10 ng/ml.

Drugs. The PI3K inhibitor LY294002, Roscovitine, the MEK1/2 inhibitorPD98059, and 10058-F4 were purchased from Sigma. The E2F inhibitorHLM006474 was purchased from Millipore. PD0332991 was purchased fromSelleckChem. JQ1 was purchased from ApexBio.

Immunostaining. Myosin heavy chain expression was detected with use ofthe MF20 antibody (R&D systems). Cells were fixed in 4% PFA,permeabilized in 0.1% Triton-X, and co-stained with antibody and DAPI(Sigma).

RNA isolation and RT-PCR. RNA was isolated using RNAeasy plus kit(Qiagen). Reverse transcription was performed using iScript™ cDNAsynthesis (Bio-rad). Real-time quantitative PCR was done on a CFX96™ PCRmachine using SYBR green supermix (Bio-rad). A complete list of primersused is provided in the Supplementary Methods.

Microarray analysis. RNA time-course samples were hybridized to IlluminaRef8 BeadChip™ arrays. Data analysis was performed with GenomeStudio™software and the help of the BCH IDDRC Molecular Genetics Core.

Cloning. The mouse p21 open reading frame was cloned into thepmCherry-N1™ plasmid (Clontech) with a self-cleaving P2A peptide andmCherry fused to its C-terminus. The plasmid was transfected into theMyoD-inducible ES cell line and selected by G418. The final line wasderived from the picking of a single cell clone colony.

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References Relevant to Schematics Presented in FIGS. 1A-1C and 7A-7C

-   Leone G, et al. (2001) Myc requires distinct E2F activities to    induce S phase and apoptosis. Mol. Cell. 8:105-113).-   Lasorella, A., Noseda, M., Beyna, M., Yokota, Y. & Iavarone, A. Id2    is a retinoblastoma protein target and mediates signalling by Myc    oncoproteins. Nature 407, 592-598 (2000)-   La Rocca S A, Crouch D H, Gillespie D (1994) c-Myc inhibits myogenic    differentiation and myoD expression by a mechanism which can be    dissociated from cell transformation. Oncogene 9:3499-508.-   Philipp A, et al. (1994) Repression of cyclin D1: a novel function    of Myc? Mol. Cell Biol. 14:4032-4043.-   Watanabe G, et al. (1998) Inhibition of cyclin D1 kinase activity is    associated with E2F-mediated inhibition of cyclin D1 promoter    activity through E2F and Sp1. Mol. Cell Biol. 18:3212-3222.-   Jirmanova L, et al. (2002) Differential contributions of ERK and    PI3-kinase to the regulation of cyclin D1 expression and to the    control of the G1/S transition in mouse embryonic stem cells.    Oncogene 21:5515-5528.-   Oswald F, et al. (1994) E2F-dependent regulation of human Myc:    transactivation by Cyclin D1 and Cyclin A overrides tumour    suppressor protein functions. Oncogene 9:2029-2036.-   Tintignac L A et al. (2000) Cyclin E-cdk2 phosphorylation promotes    late G1-phase degradation of MyoD in muscle cells. Experimental Cell    Research 259:300-307-   Lassar A B, Skapek S X, and Novitch B (1994) Regulatory mechanisms    that coordinate skeletal muscle differentiation and cell cycle    withdrawal. Curr. Opin. Cell Biol. 6:788-794.-   Kitzmann M, and Fernandez A (2001) Crosstalk between cell cycle    regulators and the myogenic factor MyoD in skeletal myoblasts. Cell.    Mol. Life Sci. 58:571-579.-   Cartwright P, et al. (2005) LIF/STAT3 controls ES-cell self-renewal    and pluripotency by a Myc-dependent mechanism. Development    132:885-96.-   Paling N R D, Wheadon H, Bone H K and Welham M J. (2005) Regulation    of embryonic stem cell self-renewal by phosphoinositide 3-kinase    dependent signalling. J. Biol. Chem. 279:48063-48070.-   Ying Q L, Nichols J, Chambers I, Smith A (2003) BMP induction of Id    proteins suppresses differentiation and sustains embryonic stem cell    self-renewal in collaboration with STAT3. Cell 115:281-292.-   Sakamuro D and Prendergast G C (1999) New Myc-interacting proteins:    a second Myc network emerges. Oncogene 18:2942-2954.

TABLE 2 qRT-PCR Primer list. Primers are labelled (Genename_)(F or R) depending on forward or reverse Primer Primer SequenceSEQ ID NO GAPDH_F GTGTTCCTACCCCCAATGTGT 01 GAPDH_R GTTGAAGTCGCAGGAGACAAC02 NeuroD1_F ACAGACGCTCTGCAAAGGTTT 03 NeuroD1_R GGACTGGTAGGAGTAGGGATG 04Pax6_F TACCAGTGTCTACCAGCCAAT 05 Pax6_R TGCACGAGTATGAGGAGGTCT 06 Dll1_FGCAGGACCTTCTTTCGCGTAT 07 Dll1_R AAGGGGAATCGGATGGGGTT 08 Pparg_FTTTTCCGAAGAACCATCCGATT 09 Pparg_R ATGGCATTGTGAGACATCCCC 010 Nkx2.2_FAAGCATTTCAAAACCGACGGA 011 Nkx2.2_R CCTCAAATCCACAGATGACCAGA 012 Ngn3_FCCAAGAGCGAGTTGGCACT 013 Ngn3_R CGGGCCATAGAAGCTGTGG 014 CEBPa_FGATAAGAACAGCAACGAGTACCG 015 CEBPa_R GTCACTGGTCAACTCCAACACC 016 CEBPb_FCATCGACTTCAGCCCCTACC 017 CEBPb_R GGCTCACGTAACCGTAGTCG 018 MeF2c_FTCTCCGCGTTCTTATCCCAC 019 MeF2c_R AGGAGTTGCTACGGAAACCAC 020 Pdx1_FCCCCAGTTTACAAGCTCGCT 021 Pdx1_R CTCGGTTCCATTCGGGAAAGG 022 Nestin_FCCCTGAAGTCGAGGAGCTG 023 Nestin_R CTGCTGCACCTCTAAGCGA 024 Sox6_FTCAACCTGCCAAACAAAAGC 025 Sox6_R GCTGGATCTGTTCTCGCATC 026 Smyd1_FTCCGAGGGTTTGTATCACGAG 027 Smyd1_R CCTCCTGGCATAATGTGAGGC 028 Runx2_FCCAAGGAACAAACCGTCAAA 029 Runx2_R AAGCGGGTCTGCAGAGTGTA 030 EBF_FAGATTCCAGGTCGTGGTGTCTA 031 EBF_R ACAGGGAGTAGCATGTTCCAGA 032 Tal1_FGGTCCTCACACCAAAGTAGTGC 033 Tal1_R CGGAGGATCTCATTCTTGCTTA 034 Gata4_FATAATCTCCTTCACCCCAGCTC 035 Gata4_R GGGCAGGGCTTCTATGTCTAGT 036 Mrg1_FATTTGTTGGCTGCATGATCTTT 037 Mrg1_R CGGTATGACTTTTCCTGATCCA 038 Mitf_FAGGCAGAAAAAGGACAATCACA 039 Mitf_R CTTCCGGATGTAGTCCACAGAG 040 Gata2_FAAGCGAAAACCAAACTGCATAA 041 Gata2_R CCAAGAACCACTCAAAGGACTG 042 Gata3_FTCCCATTTGTGAATAAGCCATT 043 Gata3_R TCCTTCATGCCTTTCTTACAGC 044 Runx1_FCTCCCAATAGCCCTTCTCACTT 045 Runx1_R AGCAAGAGAATGGCTGACTCAC 046 PU1_FACAGATGCACGTCCTCGATACT 047 PU1_R CTTCTCCATCAGACACCTCCAG 048 Sox9_FTCTCCCCCTTTTCTTTGTTGTT 049 Sox9_R ACGCACACATCCACATACAGTC 050 Lbh_FCTTGCTTCCACTCTGCTCTGTT 051 Lbh_R ACGGCAAGACCAAGACAGATAA 052 Gata1_FCAGAATAGCCTTGACCTTGTGG 053 Gata1_R AGGAAAATGTCAGGCATAGCAA 054 Ikaros_FGTTTGTTGCCCAGTAAGACGAG 055 Ikaros_R GCTTTGGCTTCCAAGAAGTTTT 056 Gata6_FCCAAATCATGTGCTTCTTGTGA 057 Gata6_R TATTCTTGTTGAGACCCCAGGA 058 Oct4_FATGGCATACTGTGGACCTC 059 Oct4_R AGCAGCTTGGCAAACTGTTC 060 Nanog_FCAGCAGATGCAAGAACTCTCC 061 Nanog_R GGATACTCCACTGGTGCTGAG 062 Brachyury_FCTCTAATGTCCTCCCTTGTTGCC 063 Brachyury_R TGCAGATTGTCTTTGGCTACTT 064Sox17_F ACAACGCAGAGCTAAGCAAGAT 065 Sox17_R GTACTTGTAGTTGGGGTGGTCCT 066Cdx2_F AAAAGACAAATACCGGGTGGTG 067 Cdx2_R TGATTTTCCTCTCCTTGGCTCT 068Pax3_F CATCCGACCTGGTGCCATC 069 Pax3_R ATTTCCCAGCTAAACATGCCC 070 Pax7_FTGGGGTCTTCATCAACGGTC 071 Pax7_R ATCGGCACAGAATCTTGGAGA 072 Myf5_FCACCACCAACCCTAACCAGAG 073 Myf5_R AGGCTGTAATAGTTCTCCACCTG 074 MyoG_FGCAGGCTCAAGAAAGTGAATGA 075 MyoG_R TAGGCGCTCAATGTACTGGAT 076 MRF4_FAGAGGGCTCTCCTTTGTATCC 077 MRF4_R CTGCTTTCCGACGATCTGTGG 078 MyoD1endo_FCTGCAGCAGCAGAGGGCGCACCA 079 MyoD1endo_R GAAGAACGGCTTCGAAAGGACAGTTGG 080Six1_F GAAAGGGAGAACACCGAAAACA 081 Six1_R GTGGCCCATATTGCTCTGGA 082 Six4_FACCCCAGTACCGAGGATGAAT 083 Six4_R AACTCCAGACGAGCTTAGTGA 084

Table 3: List of factors affected by cell cycle inhibition duringunguided differentiation and the cell type lineages in which they play arole. Lineages refer to differentiation systems in which the factorshave been reported to be involved. The effect of low serum compared tohigh serum is listed as increased, decreased, or same/similar. Over theseven day time course, when the effect of low serum occurs is referredto as the stage. If the effect occurs within Days 1-3, the stage isreferred to as early, or else mid (Days 3-4), or late (Days 5-7).

Gene Lineage Increase or Decrease Stage NeuroD1 Neural Increase EarlyDll1 Neural Increase Mid Pax6 Neural Increase Mid Nestin Neural DecreaseMid/Late Sox6 Cardiac Increase Late Smyd1 Cardiac Increase Late Mef2cCardiac Increase Late Gata4 Cardiac Increase Mid/Late Gata6 CardiacIncrease Mid/Late C/EBPβ Adipocyte Same N/A PPARγ Adipocyte IncreaseLate CEBPα Adipocyte Increase Late Sox17 Pancreatic Increase Late Nkx2.2Pancreatic Increase Late Pdx1 Pancreatic Same N/A Ngn3 PancreaticDecrease Mid/Late Runx2 Osteoblast Increase Late Cdx2 Placental Same N/ACited2 Multiple Increase Late Mitf Melanocyte Increase Late Sox9Chondrocyte Increase Mid/Late Lbh Chondrocyte Same N/A Runx1 BloodIncrease Late Pu.1 Blood Decrease Early Gata2 Blood Increase Mid/LateGata3 Blood Increase Late Tal1 Blood Decrease Late EBF Blood Altered N/AGata1 Blood Increase Early to late Ikaros Blood Similar N/A

Example 2: The Generality of Direct Embryonic Stem Cell Programming

Described above herein is a principle for the direct programming of cellfates. Further described herein is a demonstration that this programmingprinciple can be extended from skeletal muscle to spinal motor neurons,cardiomyocytes, and hepatoblast-like cells. The broad applicability ofthis principle to these cell types indicates a common differentiationstructure is shared across multiple lineages, and indicates that cellcycle-associated processes serve a fundamental role in regulating therate and path of embryonic differentiation.

The induction of cell fates during embryogenesis is orchestrated throughthe action of developmental signals, such as growth factors (1). Properexposure to these signals leads to the normal patterning and growth ofthe embryo. It is described herein that the major point of thissignaling, with respect to differentiation, is to align two propertieswithin the cell: 1. its transcriptional state, which determines thelineage and cell type to be specified, and 2. its appropriate “cellcycle” state, which determines the rate of differentiation (2). Giventhe alignment of these two properties, it can be possible to inducecells to turn into any fate, even through transformations that do notoccur naturally. This decomposition of the normal differentiationprocess helps us understand the critical elements required for cell fatechanges and the roles they play, which cannot be understood by onlystudying normal embryonic differentiation. Moreover, it could improveour ability to alter cell fates for regenerative medicine

Described herein are methods relating to the combination of theappropriate transcriptional state with the appropriate “cell cycle”state to determine a specific cell fate. Neither of these two componentsis sufficient alone. However, when combined together, these twoconditions lead to a rapid and direct conversion. This was firstdemonstrated in experiments where mouse embryonic stem (ES) cells arerapidly and effectively converted into skeletal muscle. Normally, astandard strategy of differentiating ES cells into skeletal muscle wouldrequire sequential treatment of growth factors to recapitulateintermediate embryonic progenitor stages, such as mesoderm (3). Thisneed to reconstitute a series of intermediate stages can be avoided byengineering the ES cell to overexpress MyoD (the transcriptionalcomponent, which by itself does not trigger muscle differentiation) andsimultaneously using methods of inhibiting the cell cycle (such asgrowth factor withdrawal)(2). Out of the usual myogenic regulatoryhierarchy, which includes Six1, Six4, Pax3, Pax7, MyoG, Myf5, and Mrf4,only Pax3 and MyoG appear to be significantly upregulated in addition tothe exogenous MyoD.

Setting the transcriptional state can be achieved by overexpressing anappropriate transcription factor. Setting the “cell cycle” state can beachieved by manipulating cell cycle pathways. The pathways that normallyconnect extracellular growth factors to the intracellular G1/S machineryand their nuclear effectors (e.g Myc, Rb) are highly involved(summarized in network models in ref 2). Withdrawing growth factorsdelivers a strong inhibitory signal, whereas inhibiting downstreampathways has effects that are differentiation stage-specific. Theskeletal muscle system has been a model for cell differentiation, andinitial studies were performed on this system.

Described herein is the demonstration that other lineages share asimilar capacity to be directly programmed. MyoD was replaced withtranscription factors for three additional cell types. The resultsindicate a much greater generality of the process of direct programming.

Results

Spinal Motor Neurons

To generate spinal motor neurons, a mouse ES cell line thatoverexpressed a combination of three transcription factor lineagespecifiers (Ngn2, Isl1, Lhx3) was used. This combination was previouslyshown to specify spinal motor neuron identity (4). These iNIL cells, asthey are also called, upon doxycycline stimulation express the threetranscription factors from a single open reading frame coupled by 2Apeptides. Growth factor withdrawal was used as the method of cell cyclemanipulation in the experiments described in this Example.

As expected from the results on skeletal muscle myotubes, neuronsrapidly formed from ES cells upon switching to different types of growthfactor-free media. Four days after removing LIF and adding doxycycline(Day 0), ES cells that had been differentiated in growth factor-free(N2B27 or 20% Knockout Serum Replacement/KOSR) or growth factor-reducedmedia (2% horse serum/HS with insulin) expressed much higher levels ofthe terminal neuron differentiation marker beta-3 tubulin (Tubb3) thanin high growth factor media (15% FBS), and displayed morphologicalcharacteristics of neurons, which was not seen in high serum (FIG. 11Aand FIG. 14). Tubb3 expression was confirmed by immunostaining. Wheneither of the ES cell growth factors LIF or Bmp4 were added back intogrowth factor-free media, there was a strong block on neuronaldifferentiation.

To see which genes are induced during this rapid differentiationprocess, the mRNA expression of neural lineage genes was profiled acrossthe time course of differentiation (first 5 days). As before, LIF wasremoved, the media changed to N2B27, and doxycycline added at Day 0.Many neural genes were upregulated in the growth factor-free N2B27 media(FIG. 11B). These include Pax6, which had a sustained activation thatincreased during the five days of differentiation. Hb9 and NeuroD1 werealso activated, as were endogenous Lhx3 and Lhx4. Other genes that weresimilarly activated include D111, Onecut1, Onecut2, Mef2c, and Pax3.Some of these genes were upregulated even at day one. In contrast, mostof the activation of terminal genes occurred slightly later, around day2-3. These include Tubb3, neuronal guidance molecule Slit2a, axonguidance receptors Robot and Robo2, receptor Dcc, and cholinergicreceptor Chrnb2. Similar increases were not observed under the highserum condition.

Cardiomyocytes

Similar experiments were performed on an ES cell line that overexpressedthe transcription factor Gata5. The Gata4/5/6 family of transcriptionfactors have been shown to specify cardiomyocytes previously (5). Theearliest terminal marker expression for cardiomyocytes in growth factormedia could be detected by day 3 after LIF removal and dox addition.Similar to neurons, ES cells differentiated in growth factor-free orgrowth factor-reduced media displayed higher levels of genes involvedwith cardiomyocyte terminal differentiation, such as cardiac troponin(Tnnt2) by day 4, compared with high serum (FIG. 12A). The earliestspontaneously beating cardiomyocyte clusters could be observed at day 5,although this is a rare event. More commonly, beating clusters could beobserved starting at day 8-9 (FIG. 15). This may reflect a requirementfor aggregation.

Several cardiac lineage genes in N2B27 media were upregulated (FIG.12B). These include the lineage specifiers Sox6, Smyd1, Isl1, and Mef2c,as well as Fabp3, Mef2a, Tbx20, c-kit, Srf, and the Gata5 related familymembers 4 and 6. Terminal differentiation genes were also upregulatedwith rapid kinetics, including natriuretic peptide A, cardiac troponin,smooth muscle actin (Acta2), Myosin light chains 2 and 7 (Myl2/7),alpha-actinin (Actn2), and the potassium channel Hcn4, all rose within3-5 days.

Hepatoblast-Like Cells

One essential lineage specifying factor for hepatocytes is hepaticnuclear factor 4α (Hnf4α)(6). Switching of Hnf4α-overexpressing ES cellsto growth factor-free or growth factor-reduced media led to theactivation of a subset of hepatic genes. At day 0, LIF and doxycyclinewere removed and the media was either switched or kept to 15% FBS. Byday 4, there were high levels of alphafetoprotein (AFP) that could bedetected in the cells differentiated in the growth factor-free or growthfactor-reduced media and less than 10% the level of AFP in cells in 15%FBS (FIG. 13A and FIG. 16). AFP expression could be confirmed byimmunostaining. While this activation by growth factor withdrawal wassimilar to the pattern seen in previous cell types, there was anadditional interesting point of variation compared to other lineagesexamined so far. The re-addition of Bmp4 did not reduce the amount ofAFP expression, although the re-addition of LIF did, as usuallyobserved. Hnf4α-overexpressing cells continued to divide for severaldays even after incubation in growth factor-free N2B27 media. The AFPexpression and continuous division indicate that the cells havecharacteristics of hepatoblasts, rather than hepatocytes. Dexamethasone(a corticosteroid) often helps maintain hepatocyte survival in culture.However, it did not have any additional effect on AFP expression. Manyhepatic genes upregulated, including acetylcholinesterase (AchE),endogenous Hnf4α (from days 5-7), Foxa2 (from days 2-5), Foxa1, Asgr1,and Prox1 (FIG. 13B). Albumin production was also detected, as wasSox17, Pparα, Cdh3, Tbx3, Krt18, Hhex, Lrh1, C/EBPα, Hnf1a, and Hnf1b.

Discussion

The results presented herein from skeletal muscle, spinal motor neurons,cardiomyocytes, and hepatocytes show that the effects of perturbing thecell cycle after establishing the proper transcriptional state occurs inmore than one cell lineage. For spinal motor neurons and cardiomyocytes,the differentiation appeared to be more complete, including bothhistological and molecular features. The hepatocytes were less complete,and resembled hepatoblast-like cells. However, in each case directprogramming induced characteristic genes in each lineage.

The ability to directly program cell types suggests an underlying logicof how cell types are normally specified. In normal embryonicdevelopment, and in stem cell protocols that seek to mimic this process,proliferation and differentiation are usually simultaneously specifiedby growth factors. Sequential changes to the composition andconcentration of these growth factors specify cell type identity andmaintain an expanding embryo. These changes generate numerousintermediate cell types, until cell cycle exit drives cells intoterminal differentiation. In contrast, removing growth factors at anearly stage can shortcut cells into adopting late, terminal fates. Inthis sense, proliferative signals may serve as a general “rate-limitingstep” in everyday differentiation. For practical applications, thedirect programming process described herein provides an alternative tocurrent strategies of embryonic stem cell differentiation, which seek tomimic the natural differentiation pathways.

Materials and Methods

ESC culture and cell lines. ESCs were cultured in standard media (DMEMwith LIF+15% fetal bovine serum) on 0.1% gelatin-coated dishes. TheTet-Off Hnf4α line was cultured with 0.2 μg/ml doxycycline.

ESC differentiation. Twenty-four hours before starting, ESCs aretrypsinized and spread out as monolayer onto gelatin in standard ESmedia. At day 0 (ESCs), the media was switched from standard ES media toeither N2B27 media (Invitrogen), 20% Knockout Serum Replacement(KOSR)(Invitrogen), 2% horse serum (HS)(Invitrogen)+10 μg/ml insulin, orjust 15% FBS without LIF. Doxycycline was also added at day 0 (3 μg/ml)to induce expression or removed entirely for the Hnf4α line. Media wasrefreshed daily.

For the neurons, the ES cells were seeded onto plates pre-coated with amix of poly-d-lysine (100 μg/ml) and laminin (50 μg/ml) instead ofgelatin for adherence. 1000 U/ml of LIF or 10 ng/ml Bmp4 was added whenneeded. Dexamethasone (Sigma) was used at 0.1 μM.

RNA isolation and qPCR. RNA was isolated using Qiagen Rnaeasy™ Plus Kit.Purified RNA was then reverse transcribed using Bio-rail's iSCRIPT™ cDNAsynthesis kit. Quantitative PCR was performed using Bio-rad's SYBR™green Supermix.

Immunostaining Antibodies for Tubb3 and AFP were provided respectivelyby Cell Signaling (D71G9), Sino Biological (clone 27). Cells were fixedin 4% PFA and permeabilized with 0.1% Triton.

REFERENCES

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1. A method of differentiating a stem cell, the method comprising: i)contacting the stem cell with one or more ectopic differentiationfactors; and ii) inhibiting the cell cycle of the stem cell; whereinsteps i) and ii) occur within 15 days of each other. 2.-15. (canceled)16. The method of claim 1, wherein steps i) and ii) occursimultaneously.
 17. The method of claim 1, wherein the differentiationfactor is a terminal transcription factor.
 18. The method of claim 17,wherein the terminal transcription factor is selected from Table
 1. 19.The method of claim 17, wherein the stem cell is to be differentiated toa skeletal muscle phenotype and is contacted with the terminaltranscription factor MyoD.
 20. The method of claim 17, wherein the stemcell is to be differentiated to a spinal motor neuron phenotype and iscontacted with a terminal transcription factor selected from the groupconsisting of: Ngn2; Isl1; and Lhx3.
 21. The method of claim 17, whereinthe stem cell is to be differentiated to a spinal motor neuron phenotypeand is contacted with the terminal transcription factors Ngn2; Isl1; andLhx3.
 22. The method of claim 17, wherein the stem cell is to bedifferentiated to a cardiomyocyte phenotype and is contacted with theterminal transcription factor Gata5.
 23. The method of claim 17, whereinthe stem cell is to be differentiated to a hepatocyte or hepatoblastphenotype and is contacted with the terminal transcription factor Hnf4α.24. The method of claim 1, wherein the cell cycle is inhibited by one ormore of the following: reducing or removing growth factors; reducingserum levels; reducing serum levels below 5%; contacting the cell with aPI3K inhibitor; contacting the cell with an E2F family transcriptionfactor inhibitor; contacting the cell with a Myc inhibitor; contactingthe cell with a MAPK inhibitor; contacting the cell with a MEK1/2inhibitor; contacting the cell with a CDK inhibitor; contacting the cellwith an Id inhibitor; contacting the cell with a Rb agonist; contactingthe cell with a Ink family agonist; contacting the cell with a Cip/Kipfamily agonist; and culturing the cell in a media lacking a factorselected from the group consisting of: LIF; Bmp; Fgf; Activin; or TGFβ.25.-27. (canceled)
 28. The method of claim 24, wherein the PI3Kinhibitor is LY294002.
 29. The method of claim 24, wherein the E2Ftranscription factor inhibitor is HLM006474.
 30. The method of claim 24,wherein the Myc inhibitor is JQ1 or 10058-F4.
 31. The method of claim24, wherein the MAPK inhibitor is PD98059.
 32. The method of claim 24,wherein the CDK inhibitor is a CDK4 or CDK2 inhibitor.
 33. The method ofclaim 24, wherein the CDK inhibitor is p16, p15, p18, p19, p21, p27, orp57.
 34. (canceled)
 35. The method of claim 1, wherein the stem cell isan embryonic stem cell.
 36. The method of claim 1, wherein steps i) andii) result in a population of cells comprising one or moreterminally-differentiated cell types.
 37. The method of claim 1, whereinsteps i) and ii) result in a population of cells comprising no more than2 terminally-differentiated cell types.
 38. The method of claim 1,wherein steps i) and ii) result in a population of cells of which atleast 50% are terminally-differentiated cells. 39.-41. (canceled)